Glucoamylase variants and polynucleotides encoding same

ABSTRACT

The present invention relates to glucoamylase variants having improved thermostability. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/EP2012/070127 filed Oct. 11, 2012, which claims priority or thebenefit under 35 U.S.C. 119 of U.S. provisional application No.61/545,628 filed Oct. 11, 2011, the contents of which are fullyincorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to glucoamylase variants, polynucleotidesencoding the variants, methods of producing the variants, and methods ofusing the variants.

2. Description of the Related Art

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is anenzyme, which catalyzes the release of D-glucose from the non-reducingends of starch or related oligo- and polysaccharide molecules.Glucoamylases are produced by several filamentous fungi and yeast, withthose from Aspergillus being commercially most important.

Commercially, glucoamylases are used to convert starchy material, whichis already partially hydrolyzed by an alpha-amylase, to glucose. Theglucose may then be converted directly or indirectly into a fermentationproduct using a fermenting organism. Examples of commercial fermentationproducts include alcohols (e.g., ethanol, methanol, butanol,1,3-propanediol); organic acids (e.g., citric acid, acetic acid,itaconic acid, lactic acid, gluconic acid, gluconate, lactic acid,succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone);amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂), and morecomplex compounds, including, for example, antibiotics (e.g., penicillinand tetracycline); enzymes; vitamins (e.g., riboflavin, B₁₂,beta-carotene); hormones, and other compounds which are difficult toproduce synthetically. Fermentation processes are also commonly used inthe consumable alcohol (e.g., beer and wine), dairy (e.g., in theproduction of yogurt and cheese) industries.

The end product may also be syrup. For instance, the end product may beglucose, but may also be converted, e.g., by glucose isomerase tofructose or a mixture composed almost equally of glucose and fructose.This mixture, or a mixture further enriched with fructose, is the mostcommonly used high fructose corn syrup (HFCS) commercialized throughoutthe world.

It is an object of the present invention to provide polypeptides havingglucoamylase activity and polynucleotides encoding the polypeptides andwhich provide a high yield in fermentation product production processes,such as ethanol production processes, including one-step ethanolfermentation processes from un-gelatinized raw (or uncooked) starch.Copending patent application, WO2011/127802, discloses a wild typeglucoamylase from Penicillium oxalicum.

The present invention provides a glucoamylase variant with improvedproperties compared to its parent.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to glucoamylasevariants, comprising a substitution or deletion at one or more positionscorresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 18, 26,31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112, 161, 172, 218, 220, 221,245, 253, 255, 279, 325, 327, 359, 364, 370, 375, 377, 405, 445, 447,460, 463, 465, 468, 477, 501, 502, 504, 516, 524, 526, 563, 564, 568,571 of the polypeptide of SEQ ID NO: 3, wherein the variant hasglucoamylase activity.

In a second aspect the present invention relates to a variantglucoamylase catalytic domain comprising a substitution at one or morepositions corresponding to positions 10, 11, 12, 18, 26, 31, 33, 34, 65,72, 74, 79, 80, 103, 105, 112, 161, 172, 218, 220, 221, 245, 253, 255,279, 325, 327, 359, 364, 370, 375, 377, 405, 445, 447, 460, 463, 465,468 of the polypeptide of SEQ ID NO: 3, wherein the variant catalyticdomain has glucoamylase activity.

In a third aspect the present invention relates to a compositioncomprising the variant polypeptide of the invention.

In further aspects the present invention also relates to isolatedpolynucleotides encoding the variants; nucleic acid constructs, vectors,and host cells comprising the polynucleotides; and methods of producingthe variants.

The present invention also relates to methods of using the polypeptidesof the invention in production of syrup and/or a fermentation product.

DEFINITIONS

Glucoamylase: The term glucoamylase (1,4-alpha-D-glucan glucohydrolase,EC 3.2.1.3) is defined as an enzyme, which catalyzes the release ofD-glucose from the non-reducing ends of starch or related oligo- andpolysaccharide molecules. For purposes of the present invention,glucoamylase activity is determined according to the procedure describedin the ‘Materials & Methods’-section herein.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, preferably at least 45%, more preferably at least 50%,more preferably at least 55%, more preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 100% of the glucoamylase activity ofthe mature polypeptide of SEQ ID NO: 2. In another embodiment thepolypeptides of the present invention have at least 20%, preferably atleast 40%, preferably at least 45%, more preferably at least 50%, morepreferably at least 55%, more preferably at least 60%, more preferablyat least 65%, more preferably at least 70%, more preferably at least75%, more preferably at least 80%, more preferably at least 85%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the glucoamylase activity of thepolypeptide of SEQ ID NO: 3.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has glucoamylaseactivity. In one aspect, a fragment contains at least 465 amino acidresidues (e.g., amino acids 30 to 494 of SEQ ID NO: 2).

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent. Suchimproved properties include, but are not limited to, improvedthermo-stability.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 22 to 616 of SEQ ID NO: 2 based on theprogram SignalP (Nielsen et al., 1997, Protein Engineering 10:1-6) thatpredicts amino acids 1 to 21 of SEQ ID NO: 2 are a signal peptide. It isknown in the art that a host cell may produce a mixture of two of moredifferent mature polypeptides (i.e., with a different C-terminal and/orN-terminal amino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving glucoamylase activity. In one aspect, the mature polypeptidecoding sequence is nucleotides 64 to 1848 of SEQ ID NO: 1 based on theSignalP (Nielsen et al., 1997, supra) that predicts nucleotides 1 to 63of SEQ ID NO: 1 encode a signal peptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and either 35%formamide, following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed three times each for 15minutes using 2×SSC, 0.2% SDS at 60° C.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent glucoamylase: The term “parent” or “parentglucoamylase” means a glucoamylase to which an alteration is made toproduce the enzyme variants of the present invention. The parent may bea naturally occurring (wild-type) polypeptide or a variant or fragmentthereof.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the—nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having glucoamylase activity. In one aspect, a subsequencecontains at least 1395 nucleotides (e.g., nucleotides 88 to 1482 of SEQID NO: 1)

Variant: The term “variant” means a polypeptide having glucoamylaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition. The variants of the present invention have at least 20%, e.g.,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 100% of the glucoamylase activityof the mature polypeptide of SEQ ID NO: 2. The variants of the presentinvention have at least 20%, e.g., at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least100% of the glucoamylase activity of the polypeptide of SEQ ID NO: 3.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Wild-type glucoamylase: The term “wild-type” glucoamylase means aglucoamylase expressed by a naturally occurring microorganism, such as abacterium, yeast, or filamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide comprisedin SEQ ID NO: 2 or the polypeptide consisting of SEQ ID NO: 3 (PE001) isused to determine the corresponding amino acid residue in anotherglucoamylase. The amino acid sequence of another glucoamylase is alignedwith the mature polypeptide disclosed as amino acids 22 to 616 of SEQ IDNO: 2 or amino acids 1-5950f SEQ ID NO: 3, and based on the alignment,the amino acid position number corresponding to any amino acid residuein the mature polypeptide disclosed in SEQ ID NO: 2 or the polypeptideof SEQ ID NO: 3 is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotherglucoamylase can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537:_39-64; Katoh and Toh, 2010,Bioinformatics 26:_1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 2 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple Alterations. Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.

Different Alterations. Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Arg170Tyr, Glu” represents a substitution of arginine at position 170with tyrosine or glutamic acid. Thus, “Tyr167Gly, Ala+Arg170Gly, Ala”designates the following variants:

-   “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”,    and “Tyr167Ala+Arg 170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated glucoamylase variants,comprising a substitution at one or more positions corresponding topositions 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 18, 26, 31, 33, 34, 65,72, 74, 79, 103, 105, 112, 161, 172, 218, 220, 221, 245, 253, 255, 279,325, 327, 359, 364, 370, 375, 377, 405, 445, 447, 460, 463, 465, 468,477, 501, 502, 504, 516, 524, 526, 563, 564, 568, 571 of the polypeptideof SEQ ID NO: 3, wherein the variant has glucoamylase activity.

The present invention also relates to isolated glucoamylase variants,comprising a deletion at one or more positions corresponding topositions 80, 502, or 504 of the polypeptide of SEQ ID NO: 3, whereinthe variant has glucoamylase activity.

The above mentioned variants having substitutions or deletions should beunderstood as encompassing all possible combinations of one or moresubstitutions or deletions at the specified positions.

Variants

The present invention provides glucoamylase variants, comprising asubstitution or deletion at one or more positions corresponding topositions 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 18, 26, 31, 33, 34, 65,72, 74, 79, 80, 103, 105, 112, 161, 172, 218, 220, 221, 245, 253, 255,279, 325, 327, 359, 364, 370, 375, 377, 405, 445, 447, 460, 463, 465,468, 477, 501, 502, 504, 516, 524, 526, 563, 564, 568, 571 of thepolypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity.

In addition to the specific substitutions or deletions in a furtherembodiment the variant is selected from the group consisting of:

a) a polypeptide having at least 65% sequence identity to thepolypeptide of SEQ ID NO: 3;

b) a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, or (ii) the full-length complement of (i);

c) a polypeptide encoded by a polynucleotide having at least 65%identity to the mature polypeptide coding sequence of SEQ ID NO: 1; and

d) a fragment of the polypeptide of SEQ ID NO: 3, which has glucoamylaseactivity.

In an embodiment, the variant has sequence identity of at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%, to the amino acid sequence of the mature parent glucoamylase.

In another embodiment, the variant has at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, such as at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the polypeptide of SEQ ID NO: 3.

In one aspect, the number of alterations in the variants of the presentinvention is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 alterations.

In another aspect, the variant is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, or (ii) the full-length complement of (i) (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

In another aspect, a variant comprises a substitution or deletion at oneor more (e.g., several) positions corresponding to positions 1, 2, 3, 4,5, 6, 7, 8, 10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103,105, 112, 161, 172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359,364, 370, 375, 377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502,504, 516, 524, 526, 563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion at twopositions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8, 10,11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112, 161, 172,218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370, 375, 377,405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516, 524, 526,563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion atthree positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7,8, 10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112,161, 172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370,375, 377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516,524, 526, 563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion atfour positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8,10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112, 161,172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370, 375,377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516, 524,526, 563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion atfive positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8,10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112, 161,172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370, 375,377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516, 524,526, 563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion at sixpositions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8, 10,11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112, 161, 172,218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370, 375, 377,405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516, 524, 526,563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion atseven positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7,8, 10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112,161, 172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370,375, 377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516,524, 526, 563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion ateight positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7,8, 10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112,161, 172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370,375, 377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516,524, 526, 563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion atnine positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8,10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112, 161,172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370, 375,377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516, 524,526, 563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion at tenpositions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8, 10,11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112, 161, 172,218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370, 375, 377,405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516, 524, 526,563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion ateleven positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7,8, 10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112,161, 172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370,375, 377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516,524, 526, 563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion attwelve positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7,8, 10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112,161, 172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370,375, 377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516,524, 526, 563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion atthirteen positions corresponding to any of positions 1, 2, 3, 4, 5, 6,7, 8, 10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112,161, 172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370,375, 377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516,524, 526, 563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion atfourteen positions corresponding to any of positions 1, 2, 3, 4, 5, 6,7, 8, 10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112,161, 172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370,375, 377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516,524, 526, 563, 564, 568, and 571.

In another aspect, a variant comprises a substitution or deletion atfifteen positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7,8, 10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112,161, 172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370,375, 377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516,524, 526, 563, 564, 568, and 571.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 1. In another aspect, the aminoacid at a position corresponding to position 1 is substituted with Ala,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Lys or Glu. In another aspect,the variant comprises or consists of the substitutions R1K or R1E of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 2. In another aspect, the aminoacid at a position corresponding to position 2 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser,Thr, Trp, Tyr, or Val, preferably with Asn. In another aspect, thevariant comprises or consists of the substitution P2N of the polypeptideof SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 3. In another aspect, the aminoacid at a position corresponding to position 3 is substituted with Ala,Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Trp or Asn. In another aspect,the variant comprises or consists of the substitutions D3W or D3N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 4. In another aspect, the aminoacid at a position corresponding to position 4 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser,Thr, Trp, Tyr, or Val, preferably with Ser or Gly. In another aspect,the variant comprises or consists of the substitutions P4S or P4G of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 5. In another aspect, the aminoacid at a position corresponding to position 5 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala or Gln. In another aspect,the variant comprises or consists of the substitutions K5A or K5Q of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 6. In another aspect, the aminoacid at a position corresponding to position 6 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Arg. In another aspect, thevariant comprises or consists of the substitution G6R of the polypeptideof SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 7. In another aspect, the aminoacid at a position corresponding to position 7 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala or Val. In another aspect,the variant comprises or consists of the substitutions G7A or G7V of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 8. In another aspect, the aminoacid at a position corresponding to position 8 is substituted with Ala,Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala or Ser. In another aspect,the variant comprises or consists of the substitutions N8A or N8S of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 10. In another aspect, the aminoacid at a position corresponding to position 10 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Trp, Tyr, or Val, preferably with Asp, Lys, or Glu. In anotheraspect, the variant comprises or consists of the substitutions T10D,T10K, or T10E of the polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 11. In another aspect, the aminoacid at a position corresponding to position 11 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser,Thr, Trp, Tyr, or Val, preferably with Asp, Phe, Ser, or Ala. In anotheraspect, the variant comprises or consists of the substitutions P11D,P11F, P11S, P11W, P11Y, P11H or P11A of the polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 12. In another aspect, the aminoacid at a position corresponding to position 12 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Tyr. In another aspect, thevariant comprises or consists of the substitution F12Y of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 18. In another aspect, the aminoacid at a position corresponding to position 18 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Asn. In another aspect, thevariant comprises or consists of the substitution E18N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 26. In another aspect, the aminoacid at a position corresponding to position 26 is substituted with Ala,Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Cys or Asn. In another aspect,the variant comprises or consists of the substitutions D26C or D26N ofthe polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 31. In another aspect, the aminoacid at a position corresponding to position 31 is substituted with Ala,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ser. In another aspect, thevariant comprises or consists of the substitution R31S of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 33. In another aspect, the aminoacid at a position corresponding to position 33 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Cys or Val. In another aspect,the variant comprises or consists of the substitutions K33C or K33V ofthe polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 34. In another aspect, the aminoacid at a position corresponding to position 34 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Tyr. In another aspect, thevariant comprises or consists of the substitution K34Y of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 65. In another aspect, the aminoacid at a position corresponding to position 65 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Trp, Tyr, or Val, preferably with Ala. In another aspect, thevariant comprises or consists of the substitution T65A of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 72. In another aspect, the aminoacid at a position corresponding to position 72 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Val. In another aspect, thevariant comprises or consists of the substitution L72V of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 74. In another aspect, the aminoacid at a position corresponding to position 74 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Asn. In another aspect, thevariant comprises or consists of the substitution E74N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 79. In another aspect, the aminoacid at a position corresponding to position 79 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, or Tyr, preferably with Ala, Gly, Ile, Leu, Lys, or Ser.In another aspect, the variant comprises or consists of thesubstitutions V79A, V79G, V79I, V79L, V79S, or V79K of the polypeptideof SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a deletion at aposition corresponding to position 80. In another aspect, the variantcomprises or consists of the deletion F80* of the polypeptide of SEQ IDNO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 103. In another aspect, theamino acid at a position corresponding to position 103 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Thr, Trp, Tyr, or Val, preferably with Asn. In another aspect,the variant comprises or consists of the substitution S103N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 105. In another aspect, theamino acid at a position corresponding to position 105 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Thr, Trp, Tyr, or Val, preferably with Pro. In another aspect,the variant comprises or consists of the substitution S105P of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 112. In another aspect, theamino acid at a position corresponding to position 112 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ser. In another aspect,the variant comprises or consists of the substitution K112S of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 161. In another aspect, theamino acid at a position corresponding to position 161 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ser. In another aspect,the variant comprises or consists of the substitution K161S of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 172. In another aspect, theamino acid at a position corresponding to position 172 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Val. In another aspect,the variant comprises or consists of the substitution 1172V of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 218. In another aspect, theamino acid at a position corresponding to position 218 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala. In another aspect,the variant comprises or consists of the substitution K218A of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 220. In another aspect, theamino acid at a position corresponding to position 220 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asn. In another aspect,the variant comprises or consists of the substitution G220N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 221. In another aspect, theamino acid at a position corresponding to position 221 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asp. In another aspect,the variant comprises or consists of the substitution K221D of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 245. In another aspect, theamino acid at a position corresponding to position 245 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, or Val, preferably with Asn. In another aspect,the variant comprises or consists of the substitution Y245N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 253. In another aspect, theamino acid at a position corresponding to position 253 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asn. In another aspect,the variant comprises or consists of the substitution Q253N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 255. In another aspect, theamino acid at a position corresponding to position 255 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Thr, Trp, Tyr, or Val, preferably with Asn. In another aspect,the variant comprises or consists of the substitution S255N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 279. In another aspect, theamino acid at a position corresponding to position 279 is substitutedwith Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asn. In another aspect,the variant comprises or consists of the substitution D279N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 325. In another aspect, theamino acid at a position corresponding to position 325 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with Thr. In another aspect,the variant comprises or consists of the substitution V325T of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 327. In another aspect, theamino acid at a position corresponding to position 327 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Trp, Tyr or Phe. Inanother aspect, the variant comprises or consists of the substitutionsQ327W, Q327Y or Q327F of the polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 359. In another aspect, theamino acid at a position corresponding to position 359 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Thr, Trp, Tyr, or Val, preferably with Asn. In another aspect,the variant comprises or consists of the substitution S359N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 364. In another aspect, theamino acid at a position corresponding to position 364 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Thr, Trp, Tyr, or Val, preferably with Pro. In another aspect,the variant comprises or consists of the substitution S364P of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 370 In another aspect, the aminoacid at a position corresponding to position 370 is substituted withAla, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val, preferably with Asn. In another aspect, thevariant comprises or consists of the substitution D370N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 375. In another aspect, theamino acid at a position corresponding to position 375 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala. In another aspect,the variant comprises or consists of the substitution 1375A of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 377. In another aspect, theamino acid at a position corresponding to position 377 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Thr, Trp, Tyr, or Val, preferably with Thr. In another aspect,the variant comprises or consists of the substitution S377T of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 405. In another aspect, theamino acid at a position corresponding to position 405 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Thr. In another aspect,the variant comprises or consists of the substitution Q405T of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 445. In another aspect, theamino acid at a position corresponding to position 445 is substitutedwith Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asn. In another aspect,the variant comprises or consists of the substitution D445N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 447. In another aspect, theamino acid at a position corresponding to position 447 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with Ser. In another aspect,the variant comprises or consists of the substitution V447S of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 460. In another aspect, theamino acid at a position corresponding to position 460 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with Ser or Thr. In anotheraspect, the variant comprises or consists of the substitution V460S orV460T of the polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 463. In another aspect, theamino acid at a position corresponding to position 463 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Trp, Tyr, or Val, preferably with Asn. In another aspect,the variant comprises or consists of the substitution T463N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 465. In another aspect, theamino acid at a position corresponding to position 465 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Thr, Trp, Tyr, or Val, preferably with Asn. In another aspect,the variant comprises or consists of the substitution S465N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 468. In another aspect, theamino acid at a position corresponding to position 468 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Ser, Thr, Trp, Tyr, or Val, preferably with Thr. In another aspect,the variant comprises or consists of the substitution P468T of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 477. In another aspect, theamino acid at a position corresponding to position 477 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Trp, Tyr, or Val, preferably with Asn. In another aspect,the variant comprises or consists of the substitution T477N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 501. In another aspect, theamino acid at a position corresponding to position 501 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Val. In another aspect,the variant comprises or consists of the substitution E501V of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionor deletion at a position corresponding to position 502. In anotheraspect, the amino acid at a position corresponding to position 502 isdeleted or substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile,Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably withThr. In another aspect, the variant comprises or consists of thealteration N502* or N502T of the polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionor deletion at a position corresponding to position 504. In anotheraspect, the amino acid at a position corresponding to position 504 isdeleted or substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val, preferably withThr. In another aspect, the variant comprises or consists of thealteration Y504* or Y504T of the polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 516. In another aspect, theamino acid at a position corresponding to position 516 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Trp, Tyr, or Val, preferably with Pro. In another aspect,the variant comprises or consists of the substitution T516P of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 524. In another aspect, theamino acid at a position corresponding to position 524 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Thr. In another aspect,the variant comprises or consists of the substitution K524T of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 526. In another aspect, theamino acid at a position corresponding to position 526 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala. In another aspect,the variant comprises or consists of the substitution G526A of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 563. In another aspect, theamino acid at a position corresponding to position 563 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Ser, Thr, Trp, Tyr, or Val, preferably with Ser. In another aspect,the variant comprises or consists of the substitution P563S of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 564. In another aspect, theamino acid at a position corresponding to position 564 is substitutedwith Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asp. In another aspect,the variant comprises or consists of the substitution N564D of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 568. In another aspect, theamino acid at a position corresponding to position 568 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Trp, Tyr, or Val, preferably with Asn. In another aspect,the variant comprises or consists of the substitution T568N of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 571. In another aspect, theamino acid at a position corresponding to position 571 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ser or Glu. In anotheraspect, the variant comprises or consists of the substitutions K571S orK571E of the polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of an alteration atpositions corresponding to positions 65 and 327, such as those describedabove.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 65 and 501, such as those describedabove.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 65 and 504, such as those describedabove.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 327 and 501, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 327 and 504, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 501 and 504, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 65, 327, and 501, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 65, 327, and 504, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 65, 501, and 504, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 327, 501, and 504, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 65, 327, 501, and 504, such asthose described above.

In another aspect, the variant comprises or consists of one or more(e.g., several) substitutions selected from the group consisting ofT65A, Q327F, E501V, Y504T, Y504*.

In a further specific aspect the variant comprises one of the followingsubstitutions or combinations of substitutions:

-   T65A; or-   Q327F; or-   E501V; or-   Y504T; or-   Y504*; or-   T65A+Q327F; or-   T65A+E501V; or-   T65A+Y504T; or-   T65A+Y504*; or-   Q327F+E501V; or-   Q327F+Y504T; or-   Q327F+Y504*; or-   E501V+Y504T; or-   E501V+Y504*; or-   T65A+Q327F+E501V; or-   T65A+Q327F+Y504T; or-   T65A+E501V+Y504T; or-   Q327F+E501V+Y504T; or-   T65A+Q327F+Y504*; or-   T65A+E501V+Y504*; or-   Q327F+E501V+Y504*; or-   T65A+Q327F+E501V+Y504T; or-   T65A+Q327F+E501V+Y504*    More specifically the variants according to the invention comprises    or consist of the below combinations of substitutions or deletions    to the polypeptide of SEQ ID NO: 3.-   E501V+Y504T;-   T65A+K161S;-   T65A+Q405T;-   T65A+Q327W;-   T65A+Q327F;-   T65A+Q327Y;-   P11F+T65A+Q327F;-   R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F;-   P2N+P4S+P11F+T65A+Q327F;-   P11F+D26C+K33C+T65A+Q327F;-   P2N+P4S+P11F+T65A+Q327W+E501V+Y504T;-   R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F;-   P11F+T65A+Q327W;-   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T;-   P11F+T65A+Q327W+E501V+Y504T;-   T65A+Q327F+E501V+Y504T;-   T65A+S105P+Q327W;-   T65A+S105P+Q327F;-   T65A+Q327W+S364P;-   T65A+Q327F+S364P;-   T65A+S103N+Q327F;-   P2N+P4S+P11F+K34Y+T65A+Q327F;-   P2N+P4S+P11F+T65A+Q327F+D445N+V447S;-   P2N+P4S+P11F+T65A+1172V+Q327F;-   P2N+P4S+P11F+T65A+Q327F+N502*;-   P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E;-   P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S;-   P2N+P4S+P11F+T65A+Q327F+S377T;-   P2N+P4S+P11F+T65A+V325T+Q327W;-   P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T;-   P2N+P4S+P11F+T65A+1172V+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T;-   P2N+P4S+P11F+D26N+K34Y+T65A+Q327F;-   P2N+P4S+P11F+T65A+Q327F+1375A+E501V+Y504T;-   P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T;-   P2N+P4S+T10D+T65A+Q327F+E501V+Y504T;-   P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T;-   K5A+P11F+T65A+Q327F+E501V+Y504T;-   P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T;-   P2N+T10E+E18N+T65A+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N;-   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A;-   P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T;-   P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T;-   P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A;-   P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*;-   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T;-   K5A+P11F+T65A+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A;-   P2N+P4S+P11F+T65A+V79A+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+V79G+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+V79I+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+V79L+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+V79S+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T;-   S255N+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or-   P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T

The variants may further comprise one or more additional substitutionsat one or more (e.g., several) other positions.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for glucoamylase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

Catalytic Domain Variants

In one embodiment the variants may consist of at least the catalyticdomain of 465 amino acids, e.g. amino acids 30 to 494 in the parentglucoamylase shown as SEQ ID NO: 2, having the substitutions and/ordeletions as described herein.

A second aspect of the present invention relates to a variantglucoamylase catalytic domain comprising a substitution at one or morepositions corresponding to positions 10, 11, 12, 18, 26, 31, 33, 34, 65,72, 74, 79, 80, 103, 105, 112, 161, 172, 218, 220, 221, 245, 253, 255,279, 325, 327, 359, 364, 370, 375, 377, 405, 445, 447, 460, 463, 465,468 of the polypeptide of SEQ ID NO: 3, wherein the variant hasglucoamylase activity.

The variant glucoamylase catalytic domain may in one embodiment beselected from the group consisting of:

(a) a catalytic domain having at least 65% sequence identity to aminoacids 30 to 494 of SEQ ID NO: 2;

(b) a catalytic domain encoded by a polynucleotide that hybridizes undermedium stringency conditions with (i) nucleotides 88 to 1482 of SEQ IDNO: 1 or (ii) the full-length complement of (i);

(c) a catalytic domain encoded by a polynucleotide having at least 65%sequence identity to (i) nucleotides 88 to 1482 of SEQ ID NO: 1; and

(d) a variant of amino acids 30 to 494 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and wherein the catalytic domain has glucoamylase activity.

In one embodiment the catalytic domain may be considered to include thelinker region from amino acids 495 to 506 of SEQ ID NO: 2. Amino acids507 to 615 of SEQ ID NO: 2 correspond to a starch binding domain.

In an embodiment, the variant glucoamylase catalytic domain has sequenceidentity of at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100%, to the amino acid sequence of the parentglucoamylase catalytic domain.

In another embodiment, the variant has at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, such as at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the catalytic domain comprised in SEQ ID NO: 2,e.g. amino acids 30 to 494 of SEQ ID NO: 2.

In another aspect, the variant glucoamylase catalytic domain is encodedby a polynucleotide that hybridizes under very low stringencyconditions, low stringency conditions, medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with (i) the catalytic domain coding sequenceof SEQ ID NO: 1, or (ii) the full-length complement of (i) or (ii)(Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.).

In an embodiment, the variant glucoamylase polypeptide has improvedthermo-stability compared to the parent enzyme.

In one embodiment, the variant catalytic domain has improvedthermostability compared to the parent catalytic domain.

Parent Glucoamylases

The parent glucoamylase may be (a) a polypeptide having at least 65%sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) apolypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, or (ii) the full-length complement of (i); or (c) apolypeptide encoded by a polynucleotide having at least 65% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1.

In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 2 of at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, which have glucoamylase activity. Inone aspect, the amino acid sequence of the parent differs by no morethan 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 2. In another aspect,the parent comprises or consists of amino acids 30 to 494 of SEQ ID NO:2.

In another aspect, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 2 containing at least 465 amino acid residues, e.g., at least470 and at least 475 amino acid residues.

In another embodiment, the parent is an allelic variant of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the parent is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, or (ii) the full-length complement of (i) or (ii) (Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold SpringHarbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encoding a parentfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) the full-length complement thereof; or (iv) asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film or any otherdetection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe isnucleotides 64 to 1848 of SEQ ID NO: 1. In another aspect, the nucleicacid probe is a polynucleotide that encodes the polypeptide of SEQ IDNO: 2; the mature polypeptide thereof; or a fragment thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 1.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

In one particular embodiment the hybrid polypeptide comprises thevariant glucoamylase catalytic domain fused to a linker and acarbohydrate binding domain.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a fungal glucoamylase. For example, the parent may bea Penicillium glucoamylase such as, e.g., a Penicillium oxalicumglucoamylase.

In another aspect, the parent is a Penicillium oxalicum, e.g., theglucoamylase of SEQ ID NO: 2 or the mature polypeptide thereof.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga variant of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xyIA and xyIB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (VIIIa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C(CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular. Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus nigerglucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a recombinant host cell of the presentinvention under conditions suitable for expression of the variant; and(b) recovering the variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods include, but are notlimited to, use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assaymay be used to determine the activity of the variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably the composition alsocomprises a carrier and/or an excipient. More preferably, thecompositions are enriched in such a polypeptide. The term “enriched”indicates that the glucoamylase activity of the composition has beenincreased, e.g., with an enrichment factor of at least 1.1. Preferably,the compositions are formulated to provide desirable characteristicssuch as low color, low odor and acceptable storage stability.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase.

In a particular embodiment the composition comprises an alpha amylaseand the polypeptide according to the invention.

In a more particular embodiment the composition further comprises aprotease.

The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae; Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptide orpolypeptide compositions of the invention. The dosage of the polypeptidecomposition of the invention and other conditions under which thecomposition is used may be determined on the basis of methods known inthe art.

Combination of Glucoamylase and Alpha-Amylase

According to this aspect of the invention a glucoamylase of theinvention may be combined with an alpha-amylase. Preferably, the ratioof acid alpha-amylase to glucoamylase is between 0.05 and 5.0 AFAU/AGU.More preferably the ratio between acid alpha-amylase activity andglucoamylase activity is at least 0.10, at least 0.15, at least 0.20, atleast 0.25, at least 0.30, at least 0.35, at least 0.40, at least 0.45,at least 0.50, at least 0.55, at least 0.60, at least 0.65, at least0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, atleast 0.95, at least 1.00, at least 1.05, at least 1.10, at least 1.20,at least 1.30, at least 1.40, at least 1.50, at least 1.60, at least1.70, at least 1.80, at least 1.85, or even at least 1.90 AFAU/AGU.However, the ratio between acid alpha-amylase activity and glucoamylaseactivity should preferably be less than 4.50, less than 4.00, less than3.50, less than 3.00, less than 2.50, or even less than 2.25 AFAU/AGU.

Above composition is suitable for use in liquefaction, saccharification,and/or fermentation process, preferably in starch conversion, especiallyfor producing syrup and fermentation products, such as ethanol.

Examples are given below of preferred uses of the polypeptidecompositions of the present invention. The dosage of the polypeptidecomposition of the invention and other conditions under which thecomposition is used may be determined on the basis of methods known inthe art.

Uses

The present invention is also directed to use of a polypeptide of thepresent invention in a liquefaction, a saccharification and/or afermentation process. The polypeptide may be used in a single process,for example, in a liquefaction process, a saccharification process, or afermentation process. The polypeptide may also be used in a combinationof processes for example in a liquefaction and saccharification process,in a liquefaction and fermentation process, or in a saccharification andfermentation process, preferably in relation to starch conversion.

In a preferred aspect of the present invention, the liquefaction,saccharification and/or fermentation process includes sequentially orsimultaneously performed liquefaction and saccharification processes.

In conventional enzymatic liquefaction process, thermostablealpha-amylase is added and the long chained starch is degraded intobranched and linear shorter units (maltodextrins), but glucoamylase isnot added. The glucoamylase of the present invention is highlythermostable, so it is advantageous to add the glucoamylase in theliquefaction process. The glucoamylase of the present invention has asynergistic effect when combined with an alpha-amylase in theliquefaction process. During conventional saccharification, the dextrinsgenerated during the liquefaction process are further hydrolyzed toproduce low molecular sugars DP1-3 that can be metabolized by fermentingorganism. The hydrolysis is typically accomplished using glucoamylases;alternatively in addition to glucoamylases, alpha-glucosidases and/oracid alpha-amylases can be used.

When applying the glucoamylase of the present invention, potentially incombination with an alpha-amylase in a liquefaction and/orsaccharification process, especially in a simultaneous liquefaction andsaccharification process, the process can be conducted at a highertemperature. By conducting the liquefaction and/or saccharificationprocesses at higher temperatures the process can be carried out in ashorter period of time or alternatively the process can be carried outusing lower enzyme dosage. Furthermore, the risk of microbialcontamination is reduced when carrying the liquefaction and/orsaccharification process at higher temperature.

Conversion of Starch-Containing Material

The present invention provides a use of the glucoamylase of theinvention for producing glucoses and the like from starch. Generally,the method includes the steps of partially hydrolyzing precursor starchusing glucoamylase of the present invention either alone or in thepresence of an alpha-amylase.

The glucoamylase of the invention may also be used in combination withan enzyme that hydrolyzes only alpha-(1,6)-glucosidic bonds in moleculescomprising at least four glucosyl residues.

In a further aspect the invention relates to the use of a glucoamylaseof the invention in starch conversion. Furthermore, the glucoamylase ofthe invention may be used in a continuous starch conversion processincluding a continuous saccharification process.

Production of Syrup, Beverage and/or Fermentation Product

Uses of the glucoamylase of the invention include conversion of starchto e.g., syrup beverage, and/or a fermentation product, includingethanol.

The present invention also provides a process of using a glucoamylase ofthe invention for producing syrup, such as glucose and the like, fromstarch-containing material. Suitable starting materials are exemplifiedin the “Starch-containing materials”—section. Generally, the processcomprises the steps of partially or totally hydrolyzingstarch-containing material (liquefaction and/or saccharification) in thepresence of the glucoamylase of the present invention alone or incombination with alpha-amylase to release glucose from the non-reducingends of the starch or related oligo- and poly-saccharide molecules.

The glucoamylase of the invention may also be used in immobilized form.This is suitable and often used for producing specialty syrups, such asmaltose syrups as well as in the raffinate stream of oligosaccharides inconnection with the production of fructose syrups, e.g., high fructosesyrup (HFS).

The glucoamylase of the present invention can also be used for producingvarious beverages, such as, but not limited to, the beverage of tomato,potato, Chinese potato, sweet potato, and/or pumpkin.

Fermentation Products

The term “fermentation product” means a product produced by a processincluding a fermentation process using a fermenting organism.Fermentation products contemplated according to the invention includealcohols (e.g., arabinitol, butanol, ethanol, glycerol, methanol,ethylene glycol, 1,3-propanediol [propylene glycol], butanediol,glycerin, sorbitol, and xylitol); organic acids (e.g., acetic acid,acetonic acid, adipic acid, ascorbic acid, citric acid,2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid,gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid,itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,oxaloacetic acid, propionic acid, succinic acid, and xylonic acid);ketones (e.g., acetone); amino acids (e.g., aspartic acid, glutamicacid, glycine, lysine, serine, and threonine); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane); acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane); an alkene (e.g. pentene, hexene, heptene, and octene);gases (e.g., methane, hydrogen (H₂), carbon dioxide (CO₂), and carbonmonoxide (CO)); antibiotics (e.g., penicillin and tetracycline);enzymes; vitamins (e.g., riboflavin, B₁₂, beta-carotene); and hormones.In a preferred aspect the fermentation product is ethanol, e.g., fuelethanol; drinking ethanol, i.e., potable neutral spirits; or industrialethanol or products used in the consumable alcohol industry (e.g., beerand wine), dairy industry (e.g., fermented dairy products), leatherindustry and tobacco industry. Preferred beer types comprise ales,stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcoholbeer, low-alcohol beer, low-calorie beer or light beer. Preferredfermentation processes used include alcohol fermentation processes,which are well known in the art. Preferred fermentation processes areanaerobic fermentation processes, which are well known in the art.

Brewing

The glucoamylases of the present invention are highly thermostable andtherefore they can be used in an industry which needs starch hydrolysisat high temperature. For example, glucoamylases of the invention can beused in a brewing industry. The glucoamylases of the invention is addedin effective amounts which can be easily determined by the skilledperson in the art.

Production of a Liquefaction, Saccharification and/or FermentationProduct

In this aspect the present invention relates to a process for producinga liquefaction, saccharification and/or fermentation product fromstarch-containing material, comprising the step of: treatingstarch-containing material with a polypeptide of the present invention.

Suitable starch-containing starting materials are listed in the“Starch-containing materials”—section below. Contemplated enzymes arelisted in the “Enzymes”—section below. Preferably the process of presentinvention comprises treating starch-containing material with apolypeptide of the present invention alone or together with analpha-amylase. The liquefaction and/or saccharification product of thepresent invention are dextrin, or low molecular sugars, for exampleDP1-3. In the liquefaction process the conversion of starch intoglucose, dextrin and/or low molecular weight sugars is enhanced by theaddition of a glucoamylase of the present invention. The fermentationproduct, such as ethanol, may optionally be recovered afterfermentation, e.g., by distillation. The fermentation is preferablycarried out in the presence of yeast, preferably a strain ofSaccharomyces. Suitable fermenting organisms are listed in the“Fermenting Organisms”—section below.

Process for Producing Fermentation Products from Gelatinized StarchContaining Material

In this aspect the present invention relates to a process for producinga fermentation product, especially ethanol, from starch-containingmaterial, which process includes a liquefaction step and sequentially orsimultaneously performed saccharification and fermentation steps.

The invention relates to a process for producing a fermentation productfrom starch-containing material comprising the steps of:

(a) liquefying starch-containing material; using an alpha amylase;

(b) saccharifying the liquefied material obtained in step (a) using aglucoamylase; and

(c) fermenting the saccharified material using a fermenting organism.

Preferably step (a) includes also using the glucoamylase of theinvention. In one embodiment the glucoamylase of the invention is alsopresent/added in step (b).

The fermentation product, such as especially ethanol, may optionally berecovered after fermentation, e.g., by distillation. Suitablestarch-containing starting materials are listed in the section“Starch-containing materials”—section below. Contemplated enzymes arelisted in the “Enzymes”—section below. The liquefaction is preferablycarried out in the presence of an alpha-amylase. The fermentation ispreferably carried out in the presence of yeast, preferably a strain ofSaccharomyces. Suitable fermenting organisms are listed in the“Fermenting Organisms”—section below. In preferred embodiments step (b)and (c) are carried out sequentially or simultaneously (i.e., as SSFprocess). In a particular embodiment, the process of the inventionfurther comprises, prior to the step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by milling; and

y) forming a slurry comprising the starch-containing material and water.

The aqueous slurry may contain from 10-40 wt. %, preferably 25-35 wt. %starch-containing material. The slurry is heated to above thegelatinization temperature and alpha-amylase, preferably bacterialand/or acid fungal alpha-amylase, may be added to initiate liquefaction(thinning). The slurry may in an embodiment be jet-cooked to furthergelatinize the slurry before being subjected to an alpha-amylase in step(a) of the invention.

More specifically liquefaction may be carried out as a three-step hotslurry process. The slurry is heated to between 60-95° C., preferably80-85° C., and alpha-amylase is added to initiate liquefaction(thinning). Then the slurry may be jet-cooked at a temperature between95-140° C., preferably 105-125° C., for 1-15 minutes, preferably for3-10 minute, especially around 5 minutes. The slurry is cooled to 60-95°C. and more alpha-amylase is added to finalize hydrolysis (secondaryliquefaction). The liquefaction process is usually carried out at pH4.5-6.5, in particular at a pH between 5 and 6. Milled and liquefiedwhole grains are known as mash.

The saccharification in step (b) may be carried out using conditionswell know in the art. For instance, a full saccharification process maylast up to from about 24 to about 72 hours, however, it is common onlyto do a pre-saccharification of typically 40-90 minutes at a temperaturebetween 30-65° C., typically about 60° C., followed by completesaccharification during fermentation in a simultaneous saccharificationand fermentation process (SSF process). Saccharification is typicallycarried out at temperatures from 30-65° C., typically around 60° C., andat a pH between 4 and 5, normally at about pH 4.5.

The most widely used process in fermentation product, especiallyethanol, production is the simultaneous saccharification andfermentation (SSF) process, in which there is no holding stage for thesaccharification, meaning that fermenting organism, such as yeast, andenzyme(s) may be added together. SSF may typically be carried out at atemperature between 25° C. and 40° C., such as between 29° C. and 35°C., such as between 30° C. and 34° C., such as around 32° C. Accordingto the invention the temperature may be adjusted up or down duringfermentation.

In accordance with the present invention the fermentation step (c)includes, without limitation, fermentation processes used to producealcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citricacid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones(e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ andCO₂); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins(e.g., riboflavin, B12, beta-carotene); and hormones. Preferredfermentation processes include alcohol fermentation processes, as arewell known in the art. Preferred fermentation processes are anaerobicfermentation processes, as are well known in the art.

Processes for Producing Fermentation Products from Un-GelatinizedStarch-Containing

In this aspect the invention relates to processes for producing afermentation product from starch-containing material withoutgelatinization of the starch-containing material (i.e., uncookedstarch-containing material). According to the invention the desiredfermentation product, such as ethanol, can be produced withoutliquefying the aqueous slurry containing the starch-containing material.In one embodiment a process of the invention includes saccharifying(milled) starch-containing material, e.g., granular starch, below thegelatinization temperature in the presence of an alpha amylase toproduce sugars that can be fermented into the desired fermentationproduct by a suitable fermenting organism. In another embodiment aglucoamylase of the invention and an alpha amylase are used duringsaccharification and fermentation. In one aspect the invention relatesto a process for producing a fermentation product from starch-containingmaterial comprising:

(a) saccharifying starch-containing material with a mature glucoamylaseaccording to the invention, preferably having the sequence shown asamino acids 22 to 616 in SEQ ID NO: 2, at a temperature below theinitial gelatinization temperature of said starch-containing material,

(b) fermenting using a fermenting organism.

Steps (a) and (b) of the process of the invention may be carried outsequentially or simultaneously. In an embodiment, a slurry comprisingwater and starch-containing material, is prepared before step (a).

In a preferred embodiment step (a) includes addition of an alphaamylase.

The fermentation process may be carried out for a period of 1 to 250hours, preferably is from 25 to 190 hours, more preferably from 30 to180 hours, more preferably from 40 to 170 hours, even more preferablyfrom 50 to 160 hours, yet more preferably from 60 to 150 hours, even yetmore preferably from 70 to 140 hours, and most preferably from 80 to 130hours.

The term “initial gelatinization temperature” means the lowesttemperature at which gelatinization of the starch commences. Starchheated in water begins to gelatinize between 50° C. and 75° C.; theexact temperature of gelatinization depends on the specific starch, andcan readily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In the context of this invention the initial gelatinizationtemperature of a given starch-containing material is the temperature atwhich birefringence is lost in 5% of the starch granules using themethod described by Gorinstein and Lii, 1992, Starch/Stärke 44(12):461-466.

Before step (a) a slurry of starch-containing material, such as granularstarch, having 10-55 wt. % dry solids, preferably 25-40 wt. % drysolids, more preferably 30-35 wt. % dry solids of starch-containingmaterial may be prepared. The slurry may include water and/or processwaters, such as stillage (backset), scrubber water, evaporatorcondensate or distillate, side stripper water from distillation, orother fermentation product plant process water. Because the process ofthe invention is carried out below the gelatinization temperature andthus no significant viscosity increase takes place, high levels ofstillage may be used if desired. In an embodiment the aqueous slurrycontains from about 1 to about 70 vol. % stillage, preferably 15-60%vol. % stillage, especially from about 30 to 50 vol. % stillage.

The starch-containing material may be prepared by reducing the particlesize, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably0.1-0.5 mm. After being subjected to a process of the invention at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or preferably at least99% of the dry solids of the starch-containing material is convertedinto a soluble starch hydrolysate.

The process of the invention is conducted at a temperature below theinitial gelatinization temperature. Preferably the temperature at whichstep (a) is carried out is between 30-75° C., preferably between 45-60°C.

In a preferred embodiment step (a) and step (b) are carried out as asequential or simultaneous saccharification and fermentation process. Insuch preferred embodiment the process is typically carried at atemperature between 25° C. and 40° C., such as between 29° C. and 35°C., such as between 30° C. and 34° C., such as around 32° C. Accordingto the invention the temperature may be adjusted up or down duringfermentation.

In an embodiment simultaneous saccharification and fermentation iscarried out so that the sugar level, such as glucose level, is kept at alow level such as below 6 wt. %, preferably below about 3 wt. %,preferably below about 2 wt. %, more preferred below about 1 wt. %, evenmore preferred below about 0.5 wt. %, or even more preferred 0.25 wt. %,such as below about 0.1 wt. %. Such low levels of sugar can beaccomplished by simply employing adjusted quantities of enzyme andfermenting organism. A skilled person in the art can easily determinewhich quantities of enzyme and fermenting organism to use. The employedquantities of enzyme and fermenting organism may also be selected tomaintain low concentrations of maltose in the fermentation broth. Forinstance, the maltose level may be kept below about 0.5 wt. % or belowabout 0.2 wt. %.

The process may be carried out at a pH in the range between 3 and 7,preferably from pH 3.5 to 6, or more preferably from pH 4 to 5.

The glucoamylase of the present invention is highly thermostable, so thepre-saccharification and/or saccharification of the present inventioncan be carried at a higher temperature than the conventionalpre-saccharification and/or saccharification. In one embodiment aprocess of the invention includes pre-saccharifying starch-containingmaterial before simultaneous saccharification and fermentation (SSF)process. The pre-saccharification can be carried out at a hightemperature (for example, 50-85° C., preferably 60-75° C.) before movinginto SSF.

Starch-Containing Materials

Any suitable starch-containing starting material, including granularstarch, may be used according to the present invention. The startingmaterial is generally selected based on the desired fermentationproduct. Examples of starch-containing starting materials, suitable foruse in a process of present invention, include tubers, roots, stems,whole grains, corns, cobs, wheat, barley, rye, milo, sago, cassava,tapioca, sorghum, rice peas, beans, or sweet potatoes, or mixturesthereof, or cereals, sugar-containing raw materials, such as molasses,fruit materials, sugar cane or sugar beet, potatoes, andcellulose-containing materials, such as wood or plant residues, ormixtures thereof. Contemplated are both waxy and non-waxy types of cornand barley.

Fermenting Organisms

“Fermenting organism” refers to any organism, including bacterial andfungal organisms, suitable for use in a fermentation process and capableof producing desired a fermentation product. Especially suitablefermenting organisms are able to ferment, i.e., convert, sugars, such asglucose or maltose, directly or indirectly into the desired fermentationproduct. Examples of fermenting organisms include fungal organisms, suchas yeast. Preferred yeast includes strains of Saccharomyces spp., inparticular, Saccharomyces cerevisiae. Commercially available yeastinclude, e.g., Red Star™/Lesaffre Ethanol Red (available from RedStar/Lesaffre, USA) FALI (available from Fleischmann's Yeast, a divisionof Burns Philp Food Inc., USA), SUPERSTART (available from Alltech),GERT STRAND (available from Gert Strand AB, Sweden) and FERMIOL(available from DSM Specialties).

Enzymes

Glucoamylase

The glucoamylase is preferably a glucoamylase of the invention. However,as mentioned above a glucoamylase of the invention may also be combinedwith other glucoamylases.

The glucoamylase may added in an amount of 0.001 to 10 AGU/g DS,preferably from 0.01 to 5 AGU/g DS, such as around 0.05, 0.1, 0.3, 0.5,1 or 2 AGU/g DS, especially 0.05 to 0.5 AGU/g DS; or 0.02-20 AGU/g DS,preferably 0.1-10 AGU/g DS.

Alpha-Amylase

The alpha-amylase may according to the invention be of any origin.Preferred are alpha-amylases of fungal or bacterial origin.

In a preferred aspect the alpha-amylase is an acid alpha-amylase, e.g.,fungal acid alpha-amylase or bacterial acid alpha-amylase. The term“acid alpha-amylase” means an alpha-amylase (E.C. 3.2.1.1) which addedin an effective amount has activity optimum at a pH in the range of 3 to7, preferably from 3.5 to 6, or more preferably from 4-5.

Bacterial Alpha-Amylases

According to the invention a bacterial alpha-amylase may preferably bederived from the genus Bacillus.

In a preferred aspect the Bacillus alpha-amylase is derived from astrain of B. licheniformis, B. amyloliquefaciens, B. subtilis or B.stearothermophilus, but may also be derived from other Bacillus sp.Specific examples of contemplated alpha-amylases include the Bacilluslicheniformis alpha-amylase (BLA) shown in SEQ ID NO: 4 in WO 99/19467,the Bacillus amyloliquefaciens alpha-amylase (BAN) shown in SEQ ID NO: 5in WO 99/19467, and the Bacillus stearothermophilus alpha-amylase (BSG)shown in SEQ ID NO: 3 in WO 99/19467. In an embodiment of the inventionthe alpha-amylase is an enzyme having a degree of identity of at least60%, preferably at least 70%, more preferred at least 80%, even morepreferred at least 90%, such as at least 95%, at least 96%, at least97%, at least 98% or at least 99% identity to any of the sequences shownas SEQ ID NOS: 1, 2, 3, 4, or 5, respectively, in WO 99/19467.

The Bacillus alpha-amylase may also be a variant and/or hybrid,especially one described in any of WO 96/23873, WO 96/23874, WO97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documentshereby incorporated by reference). Specifically contemplatedalpha-amylase variants are disclosed in U.S. Pat. Nos. 6,093,562,6,297,038 or U.S. Pat. No. 6,187,576 (hereby incorporated by reference)and include Bacillus stearothermophilus alpha-amylase (BSGalpha-amylase) variants having a deletion of one or two amino acid inposition 179 to 182, preferably a double deletion disclosed in WO1996/023873—see e.g., page 20, lines 1-10 (hereby incorporated byreference), preferably corresponding to delta(181-182) compared to thewild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467 or deletion of amino acids 179 and 180 usingSEQ ID NO: 3 in WO 99/19467 for numbering (which reference is herebyincorporated by reference). Even more preferred are Bacillusalpha-amylases, especially Bacillus stearothermophilus alpha-amylase,which have a double deletion corresponding to delta(181-182) and furthercomprise a N193F substitution (also denoted 1181*+G182*+N193F) comparedto the wild-type BSG alpha-amylase amino acid sequence set forth in SEQID NO: 3 disclosed in WO 99/19467.

The alpha-amylase may also be a maltogenic alpha-amylase. A “maltogenicalpha-amylase” (glucan 1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is ableto hydrolyze amylose and amylopectin to maltose in thealpha-configuration. A maltogenic alpha-amylase from Bacillusstearothermophilus strain NCIB 11837 is commercially available fromNovozymes A/S, Denmark. The maltogenic alpha-amylase is described inU.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, which are herebyincorporated by reference.

Bacterial Hybrid Alpha-Amylases

A hybrid alpha-amylase specifically contemplated comprises 445C-terminal amino acid residues of the Bacillus licheniformisalpha-amylase (shown as SEQ ID NO: 4 in WO 99/19467) and the 37N-terminal amino acid residues of the alpha-amylase derived fromBacillus amyloliquefaciens (shown as SEQ ID NO: 3 in WO 99/194676), withone or more, especially all, of the following substitutions:G48A+T49I+G107A+H156Y+A181T+N190F+1201F+A209V+Q264S (using the Bacilluslicheniformis numbering). Also preferred are variants having one or moreof the following mutations (or corresponding mutations in other Bacillusalpha-amylase backbones): H154Y, A181T, N190F, A209V and Q264S and/ordeletion of two residues between positions 176 and 179, preferablydeletion of E178 and G179 (using the SEQ ID NO: 5 numbering of WO99/19467).

Fungal Alpha-Amylases

Fungal acid alpha-amylases include acid alpha-amylases derived from astrain of the genus Aspergillus, such as Aspergillus oryzae, Aspergillusniger, or Aspergillus kawachii alpha-amylases.

A preferred acid fungal alpha-amylase is a Fungamyl-like alpha-amylasewhich is preferably derived from a strain of Aspergillus oryzae. In thepresent disclosure, the term “Fungamyl-like alpha-amylase” indicates analpha-amylase which exhibits a high identity, i.e. more than 70%, morethan 75%, more than 80%, more than 85% more than 90%, more than 95%,more than 96%, more than 97%, more than 98%, more than 99% or even 100%identity to the mature part of the amino acid sequence shown in SEQ IDNO: 10 in WO 96/23874.

Another preferred acid alpha-amylase is derived from a strainAspergillus niger. In a preferred aspect the acid fungal alpha-amylaseis the one from A. niger disclosed as “AMYA_ASPNG” in theSwiss-prot/TeEMBL database under the primary accession no. P56271 anddescribed in more detail in WO 89/01969 (Example 3). The acidAspergillus niger acid alpha-amylase is also shown as SEQ ID NO: 1 in WO2004/080923 (Novozymes) which is hereby incorporated by reference. Alsovariants of said acid fungal amylase having at least 70% identity, suchas at least 80% or even at least 90% identity, such as at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to SEQID NO: 1 in WO 2004/080923 are contemplated.

In a preferred aspect the alpha-amylase is derived from Aspergilluskawachii and disclosed by Kaneko et al. J. Ferment. Bioeng. 81:292-298(1996) “Molecular-cloning and determination of the nucleotide-sequenceof a gene encoding an acid-stable alpha-amylase from Aspergilluskawachii”; and further as EMBL:#AB008370.

The fungal acid alpha-amylase may also be a wild-type enzyme comprisinga carbohydrate-binding module (CBM) and an alpha-amylase catalyticdomain (i.e., a none-hybrid), or a variant thereof. In an embodiment thewild-type acid alpha-amylase is derived from a strain of Aspergilluskawachii.

An acid alpha-amylases may according to the invention be added in anamount of 0.01 to 10 AFAU/g DS, preferably 0.01 to 5 AFAU/g DS,especially 0.02 to 2 AFAU/g DS.

Fungal Hybrid Alpha-Amylases

In a preferred aspect the fungal acid alpha-amylase is a hybridalpha-amylase. Preferred examples of fungal hybrid alpha-amylasesinclude the ones disclosed in WO 2005/003311 or U.S. Patent Publicationno. 2005/0054071 (Novozymes) or US patent application No. 2006/0148054(Novozymes) which is hereby incorporated by reference. A hybridalpha-amylase may comprise an alpha-amylase catalytic domain (CD) and acarbohydrate-binding domain/module (CBM) and optional a linker.

Specific examples of contemplated hybrid alpha-amylases include, but notlimited to those disclosed in U.S. patent application No. 2006/0148054including Fungamyl variant with catalytic domain JA118 and Atheliarolfsii SBD (SEQ ID NO: 100 in U.S. application No. 2006/0148054),Rhizomucor pusillus alpha-amylase with Athelia rolfsii AMG linker andSBD (SEQ ID NO: 101 in U.S. application No. 2006/0148054) and Meripilusgiganteus alpha-amylase with Athelia rolfsii glucoamylase linker and SBD(SEQ ID NO: 102 in U.S. application No. 2006/0148054); and Rhizomucorpusillus alpha-amylase with Aspergillus niger glucoamylase linker andCBM (SEQ ID NO 2 in international publication No. WO2007/144424).

Other specific examples of contemplated hybrid alpha-amylases include,but not limited to those disclosed in U.S. Patent Publication no.2005/0054071, including those disclosed in Table 3 on page 15, such asAspergillus niger alpha-amylase with Aspergillus kawachii linker andstarch binding domain.

Commercial Alpha-Amylase Products

Preferred commercial compositions comprising alpha-amylase includeMYCOLASE from DSM (Gist Brocades), BAN™, TERMAMYL™ SC, FUNGAMYL™,LIQUOZYME™ SC, LIQUOZYME™ SC DS, and SAN™ SUPER, SAN™ EXTRA L (NovozymesA/S) and CLARASE™ L-40,000, DEX-LO™, SPEZYME™ FRED, SPEZYME™ AA,SPEZYME™ Ethyl, and SPEZYME™ DELTA AA (Genencor Int.)

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Materials and Methods

Glucoamylase Activity

Glucoamylase activity may be measured in AGU Units.

Glucoamylase Activity (AGU)

The Glucoamylase Unit (AGU) is defined as the amount of enzyme, whichhydrolyzes 1 micromole maltose per minute under the standard conditions(37° C., pH 4.3, substrate: maltose 100 mM, buffer: acetate 0.1 M,reaction time 6 minutes as set out in the glucoamylase incubationbelow), thereby generating glucose.

glucoamylase incubation: Substrate: maltose 100 mM Buffer: acetate 0.1MpH: 4.30 ± 0.05 Incubation temperature: 37° C. ± 1   Reaction time: 6minutes Enzyme working range: 0.5-4.0 AGU/mL

The analysis principle is described by 3 reaction steps:

Step 1 is an enzyme reaction:

Glucoamylase (AMG), EC 3.2.1.3 (exo-alpha-1,4-glucan-glucohydrolase),hydrolyzes maltose to form alpha-D-glucose. After incubation, thereaction is stopped with NaOH.

Steps 2 and 3 result in an endpoint reaction:

Glucose is phosphorylated by ATP, in a reaction catalyzed by hexokinase.The glucose-6-phosphate formed is oxidized to 6-phosphogluconate byglucose-6-phosphate dehydrogenase. In this same reaction, an equimolaramount of NAD+ is reduced to NADH with a resulting increase inabsorbance at 340 nm. An autoanalyzer system such as Konelab 30 Analyzer(Thermo Fisher Scientific) may be used.

Colour reaction Tris approx. 35 mM ATP 0.7 mM NAD⁺ 0.7 mM Mg²⁺ 1.8 mMHexokinase >850 U/L Glucose-6-P-DH >850 U/L pH approx. 7.8 Temperature37.0° C. ± 1.0° C. Reaction time 420 sec Wavelength 340 nmAcid Alpha-Amylase Activity

When used according to the present invention the activity of any acidalpha-amylase may be measured in AFAU (Acid Fungal Alpha-amylase Units).Alternatively activity of acid alpha-amylase may be measured in KNU-s(Kilo Novozymes Units (Termamyl SC)).

Acid Alpha-Amylase Activity (AFAU)

Acid alpha-amylase activity may be measured in AFAU (Acid FungalAlpha-amylase Units). 1 AFAU is defined as the amount of enzyme whichdegrades 5.260 mg starch dry matter per hour under the below mentionedstandard conditions.

Acid alpha-amylase, an endo-alpha-amylase(1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzesalpha-1,4-glucosidic bonds in the inner regions of the starch moleculeto form dextrins and oligosaccharides with different chain lengths. Theintensity of color formed with iodine is directly proportional to theconcentration of starch. Amylase activity is determined using reversecolorimetry as a reduction in the concentration of starch under thespecified analytical conditions.

-   -   Standard Conditions/Reaction Conditions:    -   Substrate: Soluble starch, approx. 0.17 g/L    -   Buffer: Citrate, approx. 0.03 M    -   Iodine (I₂): 0.03 g/L    -   CaCl₂: 1.85 mM    -   pH: 2.50±0.05    -   Incubation temperature: 40° C.    -   Reaction time: 23 seconds    -   Wavelength: 590 nm    -   Enzyme concentration: 0.025 AFAU/mL    -   Enzyme working range: 0.01-0.04 AFAU/mL        DNA Manipulations

Unless otherwise stated, DNA manipulations and transformations wereperformed using standard methods of molecular biology as described inSambrook et al. (1989) Molecular cloning: A laboratory manual, ColdSpring Harbor lab. Cold Spring Harbor, N.Y.; Ausubel, F. M. et al.(eds.) “Current protocols in Molecular Biology”, John Wiley and Sons,1995; Harwood, C. R. and Cutting, S. M. (eds.).

DNA Sequencing

E. coli transformation for DNA sequencing was carried out byelectroporation (BIO-RAD Gene Pulser) or chemically. DNA Plasmids wereprepared by alkaline method (Molecular Cloning, Cold Spring Harbor) orwith the Qiagen® Plasmid Kit. DNA fragments were recovered from agarosegel by the Qiagen gel extraction Kit. PCR was performed using a PTC-200DNA Engine. The ABI PRISM™ 310 Genetic Analyzer was used fordetermination of all DNA sequences.

Media

YP-2% Maltose was composed of 10 g/L yeast extract, 20 g/L pepton and 20g/L maltose.

MLC medium was composed of 40 g/L Glucose, 50 g/L Soybean powder, 4 g/LCitric acid, pH 5.0.

M410 medium was composed of 50 g/L maltose-1H₂O, 8 g/L Yeast extract, 2g/L MgSO₄.7H₂O, 4 g/L Citric acid-1H₂O, 50 g/L glucose, 2 g/L K₂HPO₄,0.5 ml/L AMG trace metal solution, and 2 g/L urea, pH4.5. AMG tracemetal solution was composed of 13.9 g/L FeSO₄.7H₂O, 13.5 g/L MnSO₄.1H₂O,6.8 g/L ZnCl₂, 2.5 g/L CuSO₄.5H₂O, 0.24 g/L NiCl₂.6H₂O, and 3 g/L citricacid.

Unless otherwise stated, media are prepared according to Sambrook et al.(1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab.,Cold Spring Harbor, N.Y. Chemicals used as buffers and substrates werecommercial products of at least reagent grade.

FT X-14 medium was composed as shown below.

Magnesiumsulfat MgSO₄, 7H₂O 0.3 g Kaliumsulfat K₂SO₄ 0.3 gNatriumchlorid NaCl 1 g Kaliumdihydrogenphosphat KH₂PO₄ 1 g MaltoseC₁₂H₂₂O₁₁•H₂O 10 g Yeast Extract 1.4 g Dimethylmalonic acid C₅H₈O₄ 10 gTrace metals 0.25 ml MSA-SUB-FS-0044 Water ad 1000 mlEnzymesGlucoamylases:

Penicillium oxalicum glycoamylase as disclosed in SEQ ID NO: 2 ofWO2011/127802.

Talaromyces emersonii glucoamylase (which is disclosed in internationalpublication WO 99/28448 as SEQ ID NO: 7)

Aspergillus niger glucoamylase (uniprot:P69328) (which is disclosed inSvensson, B. Larsen, K. Gunnarsson, A.; “Characterization of aglucoamylase G2 from Aspergillus niger.”; Eur. J. Biochem. 154:497-502(1986))

Trametes cingulata glucoamylase as disclosed in SEQ ID NO: 2 in WO2006/069289 and available from Novozymes A/S.

Alpha-Amylases:

Acid alpha-amylase disclosed as Variant JA001 in internationalpublication WO 2005/003311

Alpha-amylase produced from Bacillus licheniformis, e.g Termamyl™ SC(commercially available alpha-amylase from Novozymes A/S, Bagsvaerd,Denmark)

Hybrid alpha-amylase consisting of Rhizomucor pusillus alpha-amylasewith Aspergillus niger glucoamylase linker and SBD disclosed as V039 inTable 5 in WO 2006/069290 (Novozymes A/S)

Alpha-Amylase A: Bacillus stearothermophilus alpha-amylase (SEQ ID NO: 3in EP1023439B1) with the mutations 1181*+G182*+N193F truncated to 491amino acids shown as SEQ ID NO: 6 in WO2011/082425.

Alpha amylase 1407: Bacillus stearothermophilus alpha-amylase (SEQ IDNO: 3 in EP1023439B1) with the mutations1181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254Struncated to 491 amino acids (see also WO2011/082425).

Proteases:

Protease 196: Metallo protease derived from Thermoascus aurantiacusCGMCC No. 0670 disclosed as amino acids 1-177 in SEQ ID NO: 2 in WO2003/048353 with the following mutations:A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L (see also WO2011/072191).

Example 1 Cloning of Penicillium oxalicum Strain Glucoamylase Gene

Preparation of Penicillium oxalicum Strain cDNA.

The cDNA was synthesized by following the instruction of 3′ RapidAmplifiction of cDNA End System (Invitrogen Corp., Carlsbad, Calif.,USA).

Cloning of Penicillium oxalicum Strain Glucoamylase Gene.

The Penicillium oxalicum glucoamylase gene was cloned using theoligonucleotide primer shown below designed to amplify the glucoamylasegene from 5′ end.

Sense primer: (SEQ ID NO: 4) 5′-ATGCGTCTCACTCTATTATCAGGTG-3′

The full length gene was amplified by PCR with Sense primer and AUAP(supplied by 3′ Rapid Amplifiction of cDNA End System) by using PlatinumHIFI Tag DNA polymerase (Invitrogen Corp., Carlsbad, Calif., USA). Theamplification reaction was composed of 5 μl of 10×PCR buffer, 2 μl of 25mM MgCl₂, 1 μl of 10 mM dNTP, 1 μl of 10 uM Sense primer, 1 μl of 10 uMAUAP, 2 μl of the first strand cDNA, 0.5 μl of HIFI Taq, and 37.5 μl ofdeionized water. The PCR program was: 94° C., 3 mins; 10 cycles of 94°C. for 40 secs, 60° C. 40 secs with 1° C. decrease per cycle, 68° C. for2 min; 25 cycles of 94° C. for 40 secs, 50° C. for 40 secs, 68° C. for 2min; final extension at 68° C. for 10 mins.

The obtained PCR fragment was cloned into pGEM-T vector (PromegaCorporation, Madison, Wis., USA) using a pGEM-T Vector System (PromegaCorporation, Madison, Wis., USA) to generate plasmid AMG 1. Theglucoamylase gene inserted in the plasmid AMG 1 was sequencingconfirmed. E. coli strain TOP10 containing plasmid AMG 1 (designatedNN059173), was deposited with the Deutsche Sammlung von Mikroorganismenand Zellkulturen GmbH (DSMZ) on Nov. 23, 2009, and assigned accessionnumber as DSM 23123.

Example 2 Expression of Cloned Penicillium oxalicum Glucoamylase

The Penicillium oxalicum glucoamylase gene was re-cloned from theplasmid AMG 1 into an Aspergillus expression vector by PCR using twocloning primer F and primer R shown below, which were designed based onthe known sequence and added tags for direct cloning by IN-FUSION™strategy.

Primer F: (SEQ ID NO: 5) 5′ ACACAACTGGGGATCCACCATGCGTCTCACTCTATTATCPrimer R: (SEQ ID NO: 6) 5′ AGATCTCGAGAAGCTTAAAACTGCCACACGTCGTTGG

A PCR reaction was performed with plasmid AMG 1 in order to amplify thefull-length gene. The PCR reaction was composed of 40 μg of the plasmidAMG 1 DNA, 1 μl of each primer (100 μM); 12.5 μl of 2× ExtensorHi-Fidelity master mix (Extensor Hi-Fidelity Master Mix, ABgene, UnitedKingdom), and 9.5 μl of PCR-grade water. The PCR reaction was performedusing a DYAD PCR machine (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA) programmed for 2 minutes at 94° C. followed by a 25 cycles of 94°C. for 15 seconds, 50° C. for 30 seconds, and 72° C. for 1 minute; andthen 10 minutes at 72° C.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 1×TAE buffer where an approximately 1.9 kb PCR product band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit (GE Healthcare, United Kingdom) according tomanufacturer's instructions. DNA corresponding to the Penicilliumoxalicum glucoamylase gene was cloned into an Aspergillus expressionvector linearized with BamHI and HindIII, using an IN-FUSION™ Dry-DownPCR Cloning Kit (BD Biosciences, Palo Alto, Calif., USA) according tothe manufacturer's instructions. The linearized vector construction isas described in WO 2005/042735 A1.

A 2 μl volume of the ligation mixture was used to transform 25 μl ofFusion Blue E. coli cells (included in the IN-FUSION™ Dry-Down PCRCloning Kit). After a heat shock at 42° C. for 45 sec, and chilling onice, 250 μl of SOC medium was added, and the cells were incubated at 37°C. at 225 rpm for 90 min before being plated out on LB agar platescontaining 50 μg of ampicillin per ml, and cultivated overnight at 37°C. Selected colonies were inoculated in 3 ml of LB medium supplementedwith 50 μg of ampicillin per ml and incubated at 37° C. at 225 rpmovernight. Plasmid DNA from the selected colonies was purified usingMini JETSTAR (Genomed, Germany) according to the manufacturer'sinstructions. Penicillium oxalicum glucoamylase gene sequence wasverified by Sanger sequencing before heterologous expression. One of theplasmids was selected for further expression, and was named XYZ XYZ1471-4.

Protoplasts of Aspergillus niger MBin118 were prepared as described inWO 95/02043. One hundred μl of protoplast suspension were mixed with 2.5μg of the XYZ1471-4 plasmid and 250 microliters of 60% PEG 4000(Applichem) (polyethylene glycol, molecular weight 4,000), 10 mM CaCl₂,and 10 mM Tris-HCl pH 7.5 were added and gently mixed. The mixture wasincubated at 37° C. for 30 minutes and the protoplasts were mixed with6% low melting agarose (Biowhittaker Molecular Applications) in COVEsucrose (Cove, 1996, Biochim. Biophys. Acta 133:51-56) (1M) platessupplemented with 10 mM acetamid and 15 mM CsCl and added as a top layeron COVE sucrose (1 M) plates supplemented with 10 mM acetamid and 15 mMCsCl for transformants selection (4 ml topagar per plate). Afterincubation for 5 days at 37° C. spores of sixteen transformants werepicked up and seed on 750 μl YP-2% Maltose medium in 96 deepwell MTplates. After 5 days of stationary cultivation at 30° C., 10 μl of theculture-broth from each well was analyzed on a SDS-PAGE (Sodium dodecylsulfate-polyacrylamide gel electrophoresis) gel, Griton XT Precast gel(BioRad, CA, USA) in order to identify the best transformants based onthe ability to produce large amount of glucoamylase. A selectedtransformant was identified on the original transformation plate and waspreserved as spores in a 20% glycerol stock and stored frozen (−80° C.).

Cultivation. The selected transformant was inoculated in 100 ml of MLCmedia and cultivated at 30° C. for 2 days in 500 ml shake flasks on arotary shaker. 3 ml of the culture broth was inoculated to 100 ml ofM410 medium and cultivated at 30° C. for 3 days. The culture broth wascentrifugated and the supernatant was filtrated using 0.2 μm membranefilters.

Alpha-Cyclodextrin Affinity Gel. Ten grams of Epoxy-activated Sepharose6B (GE Healthcare, Chalfont St. Giles, U.K) powder was suspended in andwashed with distilled water on a sintered glass filter. The gel wassuspended in coupling solution (100 ml of 12.5 mg/ml alpha-cyclodextrin,0.5 M NaOH) and incubated at room temperature for one day with gentleshaking. The gel was washed with distilled water on a sintered glassfilter, suspended in 100 ml of 1 M ethanolamine, pH 10, and incubated at50° C. for 4 hours for blocking. The gel was then washed several timesusing 50 mM Tris-HCl, pH 8 and 50 mM NaOAc, pH 4.0 alternatively. Thegel was finally packed in a 35-40 ml column using equilibration buffer(50 mM NaOAc, 150 mM NaCl, pH 4.5).

Purification of Glucoamylase from Culture Broth. Culture broth fromfermentation of A. niger MBin118 harboring the glucoamylase gene wasfiltrated through a 0.22 μm PES filter, and applied on aalpha-cyclodextrin affinity gel column previously equilibrated in 50 mMNaOAc, 150 mM NaCl, pH 4.5 buffer. Unbound material was washed off thecolumn with equilibration buffer and the glucoamylase was eluted usingthe same buffer containing 10 mM beta-cyclodextrin over 3 columnvolumes.

The glucoamylase activity of the eluent was checked to see, if theglucoamylase had bound to the alpha-cyclodextrin affinity gel. Thepurified glucoamylase sample was then dialyzed against 20 mM NaOAc, pH5.0. The purity was finally checked by SDS-PAGE, and only a single bandwas found.

Example 3 Construction and Expression of a Site-Directed Variant ofPenicillium oxalicum Glucoamylase

Two PCR reactions were performed with plasmid XYZ1471-4, described inExample 2, using primers K79V F and K79VR shown below, which weredesined to substitute lysine K at position 79 from the mature sequenceto varin V and primers F-NP003940 and R-NP003940 shown below, which weredesigned based on the known sequence and added tags for direct cloningby IN-FUSION™ strategy.

Primer K79V F 18mer (SEQ ID NO: 7) GCAGTCTTTCCAATTGACPrimer K79V R 18mer (SEQ ID NO: 8) AATTGGAAAGACTGCCCG Primer F-NP003940:(SEQ ID NO: 9) 5′ ACACAACTGGGGATCCACCATGCGTCTCACTCTATTATCPrimer R-NP003940: (SEQ ID NO: 10) 5′AGATCTCGAGAAGCTTAAAACTGCCACACGTCGTTGG

The PCR was performed using a PTC-200 DNA Engine under the conditionsdescribed below.

PCR reaction system: Conditions: 48.5 micro L H2O 1 94° C. 2 min 2 beadspuRe Taq Ready-To- 2 94° C. 30 sec Go PCR Beads (Amersham bioscineces) 355° C. 30 sec 0.5 micro L X 2100 pmole/micro L Primers 4 72° C. 90 sec(K79V F + Primer R-NP003940, K79V R + 2-4 25 cycles Primer F-NP003940) 572° C. 10 min 0.5 micro L Template DNA

DNA fragments were recovered from agarose gel by the Qiagen gelextraction Kit according to the manufacturer's instruction. Theresulting purified two fragments were cloned into an Aspergillusexpression vector linearized with BamHI and HindIII, using an IN-FUSION™Dry-Down PCR Cloning Kit (BD Biosciences, Palo Alto, Calif., USA)according to the manufacturer's instructions. The linearized vectorconstruction is as described in WO 2005/042735 A1.

The ligation mixture was used to transform E. coli DH5α cells (TOYOBO).Selected colonies were inoculated in 3 ml of LB medium supplemented with50 μg of ampicillin per ml and incubated at 37° C. at 225 rpm overnight.Plasmid DNA from the selected colonies was purified using Qiagen plasmidmini kit (Qiagen) according to the manufacturer's instructions. Thesequence of Penicillium oxalicum glucoamylase site-directed variant genesequence was verified before heterologous expression and one of theplasmids was selected for further expression, and was named pPoPE001.

Protoplasts of Aspergillus niger MBin118 were prepared as described inWO 95/02043. One hundred μl of protoplast suspension were mixed with 2.5μg of the pPoPE001 plasmid and 250 microliters of 60% PEG 4000(Applichem) (polyethylene glycol, molecular weight 4,000), 10 mM CaCl₂,and 10 mM Tris-HCl pH 7.5 were added and gently mixed. The mixture wasincubated at 37° C. for 30 minutes and the protoplasts were mixed with1% agarose L (Nippon Gene) in COVE sucrose (Cove, 1996, Biochim.Biophys. Acta 133:51-56) supplemented with 10 mM acetamid and 15 mM CsCland added as a top layer on COVE sucrose plates supplemented with 10 mMacetamid and 15 mM CsCl for transformants selection (4 ml topagar perplate). After incubation for 5 days at 37° C. spores of sixteentransformants were picked up and seed on 750 μl YP-2% Maltose medium in96 deepwell MT plates. After 5 days of stationary cultivation at 30° C.,10 μl of the culture-broth from each well was analyzed on a SDS-PAGE gelin order to identify the best transformants based on the ability toproduce large amount of the glucoamylase.

Example 4 Purification of Site-Directed Po AMG Variant PE001

The selected transformant of the variant and the strain expressing thewild type Penicillium oxalicum glucoamylase described in Example 1 wascultivated in 100 ml of YP-2% maltose medium and the culture wasfiltrated through a 0.22 μm PES filter, and applied on aalpha-cyclodextrin affinity gel column previously equilibrated in 50 mMNaOAc, 150 mM NaCl, pH 4.5 buffer. Unbound materials was washed off thecolumn with equilibration buffer and the glucoamylase was eluted usingthe same buffer containing 10 mM beta-cyclodextrin over 3 columnvolumes.

The glucoamylase activity of the eluent was checked to see, if theglucoamylase had bound to the alpha-cyclodextrin affinity gel. Thepurified glucoamylase samples were then dialyzed against 20 mM NaOAc, pH5.0.

Example 5 Characterization of PE001

Protease Stability

40 μl enzyme solutions (1 mg/ml) in 50 mM sodium acetate buffer, pH 4.5,was mixed with 1/10 volume of 1 mg/ml protease solutions such asaspergillopepsinl described in Biochem J. 1975 April; 147(1):45-53. orthe commercially available product from Sigma and aorsin described inBiochemical journal [0264-6021] Ichishima yr:2003 vol: 371 iss:Pt 2 pg:541 and incubated at 4 or 32° C. overnight. As a control experiment, H₂Owas added to the sample instead of proteases. The samples were loaded onSDS-PAGE to see if the glucoamylases are cleaved by proteases.

In SDS-PAGE, PE001 only showed one band corresponding to the intactmolecule, while the wild type glucoamylase was degraded by proteases andshowed a band at lower molecular size at 60 kCa.

TABLE 1 The result of SDS-PAGE after protease treatment Wild typeglucoamylase PE001 Protease aspergillopepsin I aorsin aspergillopepsin Iaorsin control Incubation 4 32 4 32 4 32 4 32 4 temperature (° C.)intact 100% 90% 40% 10% 100% 100% 100% 100% 100% glucoamylase (ca. 70kDa) cleaved N.D. 10% 60% 90% N.D. N.D. N.D. N.D. N.D. glucoamylase (ca.60 kDa) N.D.: not detected.

Example 6 Less Cleavage During Cultivation

Aspergillus transformant of the variant and the wild type Penicilliumoxalicum glucoamylase were cultivated in 6-well MT plates containing 4×diluted YP-2% maltose medium supplemented with 10 mM sodium acetatebuffer, pH4.5, at 32° C. for 1 week.

The culture supernatants were loaded on SDS-PAGE.

TABLE 2 The result of SDS-PAGE of the culture supernatants Wild typeglucoamylase PE001 intact glucoamylase(ca. 90% 100% 70 kDa) cleavedglucoamylase 10% N.D. (ca. 60 kDa) N.D.: not detected.

The wild type glucoamylase was cleaved by host proteasaes duringfermentation, while the variant yielded only intact molecule.

Example 7 Glucoamylase Activity of Variant Compared to Parent

The glucoamylase activity measures as AGU as described above was checkedfor the purified enzymes of the wild type Penicillium oxalicum and thevariant glucoamylase.

The Glucoamylase Unit (AGU) was defined as the amount of enzyme, whichhydrolyzes 1 micromole maltose per minute under the standard conditions(37° C., pH 4.3, substrate: maltose 100 mM, buffer: acetate 0.1 M,reaction time 6 minutes).

TABLE 3 Relative specific activity AGU/mg Penicillium oxalicum wt 100%Penicillium oxalicum PE001 102% (SEQ ID NO: 3)

Example 8 Purification of Glycoamylase Variants Having IncreasedThermostability

The variants of the invention showing increased thermostability may beconstructed and expressed similar to the procedure described in Example3. All variants according to the present invention were derived from thePE001 as the parent glucoamylase, and disclosed in SEQ ID NO: 3. Afterexpression in YPM medium, variants comprising the T65A or Q327Fsubstitution was micro-purified as follows:

Mycelium was removed by filtration through a 0.22 μm filter. 50 μlcolumn material (alpha-cyclodextrin coupled to Mini-Leakdivinylsulfone-activated agarose medium according to manufacturersrecommendations) was added to the wells of a filter plate (Whatman,Unifilter 800 μl, 25-30 μm MBPP). The column material was equilibratedwith binding buffer (200 mM sodium acetate pH 4.5) by two times additionof 200 μl buffer, vigorous shaking for 10 min (Heidolph, Titramax 101,1000 rpm) and removal of buffer by vacuum (Whatman, UniVac 3).Subsequently, 400 μl culture supernatant and 100 μl binding buffer wasadded and the plate incubated 30 min with vigorous shaking. Unboundmaterial was removed by vacuum and the binding step was repeated.Normally 4 wells were used per variant. Three washing steps were thenperformed with 200 μl buffer of decreasing ionic strength added (50/10/5mM sodium acetate, pH 4.5), shaking for 15 min and removal of buffer byvacuum. Elution of the bound AMG was achieved by two times addition of100 μl elution buffer (250 mM sodium acetate, 0.1% alpha-cyclodextrin,pH 6.0), shaking for 15 min and collection of eluted material in amicrotiter plate by vacuum. Pooled eluates were concentrated and bufferchanged to 50 mM sodium acetate pH 4.5 using centrifugal filter unitswith 10 kDa cut-off (Millipore Microcon Ultracel YM-10). Micropurifiedsamples were stored at −18° C. until testing of thermostability.

Example 9 Protein Thermal Unfolding Analysis (TSA, Thermal Shift Assay)

Protein thermal unfolding of the T65A and Q327F variants, was monitoredusing Sypro Orange (In-vitrogen, S-6650) and was performed using areal-time PCR instrument (Applied Biosystems; Step-One-Plus).

In a 96-well plate, 25 microliter micropurified sample in 50 mM AcetatepH4.5 at approx. 100 microgram/ml was mixed (5:1) with Sypro Orange(resulting conc.=5×; stock solution from supplier=5000×). The plate wassealed with an optical PCR seal. The PCR instrument was set at ascan-rate of 76 degrees C. pr. hr, starting at 25° C. and finishing at96° C.

Protein thermal unfolding of the E501V+Y504T variant, was monitoredusing Sypro Orange (In-vitrogen, S-6650) and was performed using areal-time PCR instrument (Applied Biosystems; Step-One-Plus).

In a 96-well plate, 15 microliter purified sample in 50 mM Acetate pH4.5at approx. 50 microgram/ml was mixed (1:1) with Sypro Orange (resultingconc.=5×; stock solution from supplier=5000×) with or without 200 ppmAcarbose (Sigma A8980). The plate was sealed with an optical PCR seal.The PCR instrument was set at a scan-rate of 76 degrees C. pr. hr,starting at 25° C. and finishing at 96° C.

Fluorescence was monitored every 20 seconds using in-built LED bluelight for excitation and ROX-filter (610 nm, emission).

Tm-values were calculated as the maximum value of the first derivative(dF/dK) (ref.: Gregory et al; J Biomol Screen 2009 14: 700.)

TABLE 4a Sample Tm (Deg. Celsius) +/− 0.4 PO-AMG (PE001) 80.3 VariantQ327F 82.3 Variant T65A 81.9

TABLE 4b Sample Tm (Deg. Celsius) +/− 0.4 Acarbose: − + PO-AMG (PE001)79.5 86.9 Variant E501V Y504T 79.5 95.2

Example 10 Thermostability Analysis by Differential Scanning Calorimetry(DSC)

Additional site specific variants having substitutions and/or deletionsat specific positions were constructed basically as described in Example3 and purified as described in Example 4. The thermostability of thepurified Po-AMG PE001 derived variants were determined at pH 4.0 or 4.8(50 mM Sodium Acetate) by Differential Scanning calorimetry (DSC) usinga VP-Capillary Differential Scanning calorimeter (MicroCal Inc.,Piscataway, N.J., USA). The thermal denaturation temperature, Td (° C.),was taken as the top of the denaturation peak (major endothermic peak)in thermograms (Cp vs. T) obtained after heating enzyme solutions inselected buffers (50 mM Sodium Acetate, pH 4.0 or 4.8) at a constantprogrammed heating rate of 200 K/hr.

Sample- and reference-solutions (approximately 0.3 ml) were loaded intothe calorimeter (reference: buffer without enzyme) from storageconditions at 10° C. and thermally pre-equilibrated for 10 minutes at20° C. prior to DSC scan from 20° C. to 110° C. Denaturationtemperatures were determined with an accuracy of approximately +/−1° C.

The isolated variants and the DSC data are disclosed in Table 5 below.

TABLE 5 DSC DSC Po-AMG Td (° C.) Td (° C.) name Mutations @ pH 4.0 @ pH4.8 PE001 (SEQ 82.1 83.4 ID NO: 3) PE167 E501V Y504T 82.1 PE481 T65AK161S 84.1 86.0 PE487 T65A Q405T 83.2 PE490 T65A Q327W 87.3 PE491 T65AQ327F 87.7 PE492 T65A Q327Y 87.3 PE493 P11F T65A Q327F 87.8 88.5 PE497R1K D3W K5Q G7V N8S T10K 87.8 88.0 P11S T65A Q327F PE498 P2N P4S P11FT65A Q327F 88.3 88.4 PE003 P11F D26C K33C T65A Q327F 83.3 84.0 PE009 P2NP4S P11F T65A Q327W 88.8 E501V Y504T PE002 R1E D3N P4G G6R G7A N8A 87.588.2 T10D P11D T65A Q327F PE005 P11F T65A Q327W 87.4 88.0 PE008 P2N P4SP11F T65A Q327F 89.4 90.2 E501V Y504T PE010 P11F T65A Q327W E501V 89.7Y504T PE507 T65A Q327F E501V Y504T 89.3 PE513 T65A S105P Q327W 87.0PE514 T65A S105P Q327F 87.4 PE515 T65A Q327W S364P 87.8 PE516 T65A Q327FS364P 88.0 PE517 T65A S103N Q327F 88.9 PE022 P2N P4S P11F K34Y T65A 89.7Q327F PE023 P2N P4S P11F T65A Q327F 89.9 D445N V447S PE032 P2N P4S P11FT65A I172V 88.7 Q327F PE049 P2N P4S P11F T65A Q327F 88.4 N502* PE055 P2NP4S P11F T65A Q327F 88.0 N502T P563S K571E PE057 P2N P4S P11F R31S K33V89.5 T65A Q327F N564D K571S PE058 P2N P4S P11F T65A Q327F 88.6 S377TPE064 P2N P4S P11F T65A V325T 88.0 Q327W PE068 P2N P4S P11F T65A Q327F90.2 D445N V447S E501V Y504T PE069 P2N P4S P11F T65A I172V 90.2 Q327FE501V Y504T PE073 P2N P4S P11F T65A Q327F 90.1 S377T E501V Y504T PE074P2N P4S P11F D26N K34Y 89.1 T65A Q327F PE076 P2N P4S P11F T65A Q327F90.2 I375A E501V Y504T PE079 P2N P4S P11F T65A K218A 90.9 K221D Q327FE501V Y504T PE085 P2N P4S P11F T65A S103N 91.3 Q327F E501V Y504T PE086P2N P4S T10D T65A Q327F 90.4 E501V Y504T PE088 P2N P4S F12Y T65A Q327F90.4 E501V Y504T PE097 K5A P11F T65A Q327F 90.0 E501V Y504T PE101 P2NP4S T10E E18N T65A 89.9 Q327F E501V Y504T PE102 P2N T10E E18N T65A Q327F89.8 E501V Y504T PE084 P2N P4S P11F T65A Q327F 90.5 E501V Y504T T568NPE108 P2N P4S P11F T65A Q327F 88.6 E501V Y504T K524T G526A PE126 P2N P4SP11F K34Y T65A 91.8 Q327F D445N V447S E501V Y504T PE129 P2N P4S P11FR31S K33V 91.7 T65A Q327F D445N V447S E501V Y504T PE087 P2N P4S P11FD26N K34Y 89.8 T65A Q327F E501V Y504T PE091 P2N P4S P11F T65A F80* 89.9Q327F E501V Y504T PE100 P2N P4S P11F T65A K112S 89.8 Q327F E501V Y504TPE107 P2N P4S P11F T65A Q327F 90.3 E501V Y504T T516P K524T G526A PE110P2N P4S P11F T65A Q327F 90.6 E501V N502T Y504*

Example 11 Thermostability Analysis by Thermo-Stress Test and pNPG Assay

Starting from one of the identified substitution variants from example10, identified as PE008, additional variants were tested by athermo-stress assay in which the supernatant from growth cultures wereassayed for glucoamylase (AMG) activity after a heat shock at 83° C. for5 min.

After the heat-shock the residual activity of the variant was measuredas well as in a non-stressed sample.

Description of Po-AMG pNPG Activity Assay:

The Penicillium oxalicum glucoamylase pNPG activity assay is aspectrometric endpoint assay where the samples are split in two andmeasured thermo-stressed and non-thermo-stressed. The data output istherefore a measurement of residual activity in the stressed samples.

Growth:

A sterile micro titer plate (MTP) was added 200 μL rich growth media (FTX-14 without Dowfax) to each well. The strains of interest wereinoculated in triplicates directly from frozen stocks to the MTP.Benchmark was inoculated in 20 wells. Non-inoculated wells with mediawere used as assay blanks. The MTP was placed in a plastic boxcontaining wet tissue to prevent evaporation from the wells duringincubation. The plastic box was placed at 34° C. for 4 days.

Assay:

50 μL supernatant was transferred to 50 μL 0.5M NaAc pH 4.8 to obtaincorrect sample pH.

50 μL dilution was transferred to a PCR plate and thermo-stressed at 83°C. for 5 minutes in a PCR machine. The remaining half of the dilutionwas kept at RT.

20 μL of both stressed and unstressed samples was transferred to astandard MTP. 20 μL pNPG-substrate was added to start the reaction. Theplate was incubated at RT for 1 h.

The reaction was stopped and the colour developed by adding 50 μL 0.5MNa₂CO₃. The yellow colour was measured on a plate reader (MolecularDevices) at 405 nm.

Buffers:

-   0.5M NaAc pH 4.8-   0.25M NaAc pH 4.8    Substrate, 6 mM pNPG:-   15 mg 4-nitrophenyl D-glucopyranoside in 10 mL 0.25 NaAc pH 4.8    Stop/Developing Solution:-   0.5M Na₂CO₃    Data Treatment:

In Excel the raw Abs405 data from both stressed and unstressed sampleswere blank subtracted with their respective blanks. The residualactivity (% res.act.=(Abs_(unstressed)−(Abs_(unstressed)−Abs_(stessed)))/Abs_(unstressed)*100%)was calculated and plotted relative to benchmark, Po-amg0008.

TABLE 6 Po-AMG name Mutations % residual activity PE008 P2N P4S P11FT65A Q327F 100 E501V Y504T PE085 P2N P4S P11F T65A S103N 127 Q327F E501VY504T PE097 K5A P11F T65A Q327F 106 E501V Y504T PE107 P2N P4S P11F T65AQ327F 109 E501V Y504T T516P K524T G526A PE130 P2N P4S P11F T65A V79A 111Q327F E501V Y504T PE131 P2N P4S P11F T65A V79G 112 Q327F E501V Y504TPE132 P2N P4S P11F T65A V79I 101 Q327F E501V Y504T PE133 P2N P4S P11FT65A V79L 102 Q327F E501V Y504T PE134 P2N P4S P11F T65A V79S 104 Q327FE501V Y504T PE150 P2N P4S P11F T65A L72V 101 Q327F E501V Y504T PE155S255N Q327F E501V Y504T 105

TABLE 7 Po-AMG name Mutations % residual activity PE008 P2N P4S P11FT65A Q327F 100 E501V Y504T PE179 P2N P4S P11F T65A E74N 108 V79K Q327FE501V Y504T PE180 P2N P4S P11F T65A G220N 108 Q327F E501V Y504T PE181P2N P4S P11F T65A Y245N 102 Q327F E501V Y504T PE184 P2N P4S P11F T65AQ253N 110 Q327F E501V Y504T PE185 P2N P4S P11F T65A D279N 108 Q327FE501V Y504T PE186 P2N P4S P11F T65A Q327F 108 S359N E501V Y504T PE187P2N P4S P11F T65A Q327F 102 D370N E501V Y504T PE192 P2N P4S P11F T65AQ327F 102 V460S E501V Y504T PE193 P2N P4S P11F T65A Q327F 102 V460TP468T E501V Y504T PE195 P2N P4S P11F T65A Q327F 103 T463N E501V Y504TPE196 P2N P4S P11F T65A Q327F 106 S465N E501V Y504T PE198 P2N P4S P11FT65A Q327F 106 T477N E501V Y504T

Example 12 Test for Glucoamylase Activity of Thermo-Stable VariantsAccording to the Invention

All of the above described variants disclosed in tables 5, 6, and 7 havebeen verified for Glucoamylase activity on culture supernatants usingthe pNPG assay described in Example 11.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The present invention is further described by the following numberedparagraphs.

-   [1] A glucoamylase variant, comprising a substitution or deletion at    one or more positions corresponding to positions 1, 2, 3, 4, 5, 6,    7, 8, 10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105,    112, 161, 172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359,    364, 370, 375, 377, 405, 445, 447, 460, 463, 465, 468, 477, 501,    502, 504, 516, 524, 526, 563, 564, 568, 571 of the polypeptide of    SEQ ID NO: 3, wherein the variant has glucoamylase activity.-   [2] The variant of paragraphs 1, selected from the group consisting    of:    -   a) a polypeptide having at least 65% sequence identity to the        polypeptide of SEQ ID NO: 3;    -   b) a polypeptide encoded by a polynucleotide that hybridizes        under low stringency conditions with (i) the mature polypeptide        coding sequence of SEQ ID NO: 1, or (ii) the full-length        complement of (i);    -   c) a polypeptide encoded by a polynucleotide having at least 65%        identity to the mature polypeptide coding sequence of SEQ ID NO:        1; and    -   d) a fragment of the polypeptide of SEQ ID NO: 3, which has        glucoamylase activity.-   [3] The variant of any of paragraphs 1-2, wherein the variant has at    least at least 65%, at least 70%, at least 75%, at least 80%, at    least 85%, at least 90%, at least 95%, at least 96%, at least 97%,    at least 98% or at least 99% sequence identity to the polypeptide of    SEQ ID NO: 3.-   [4] The variant of any of paragraphs 1-2, wherein the variant is    encoded by a polynucleotide that hybridizes under low stringency    conditions, medium stringency conditions, medium-high stringency    conditions, high stringency conditions, or very high stringency    conditions with (i) the mature polypeptide coding sequence of SEQ ID    NO: 1, or (ii) the full-length complement of (i).-   [5] The variant of any of paragraphs 1-2, wherein the variant is    encoded by a polynucleotide having at least 65%, at least 70%, at    least 75%, at least 80%, at least 85%, at least 90%, at least 95%,    at least 96%, at least 97%, at least 98%, at least 99% sequence    identity to the mature polypeptide coding sequence of SEQ ID NO: 1.-   [6] The variant of any of paragraphs 1-5, which is a variant of a    parent glucoamylase selected from the group consisting of:    -   a) a polypeptide having at least 65% sequence identity to the        polypeptide of SEQ ID NO: 3;    -   b) a polypeptide encoded by a polynucleotide that hybridizes        under low stringency conditions with (i) the mature polypeptide        coding sequence of SEQ ID NO: 1, or (ii) the full-length        complement of (i);    -   c) a polypeptide encoded by a polynucleotide having at least 65%        identity to the mature polypeptide coding sequence of SEQ ID NO:        1; and    -   d) a fragment of the mature polypeptide of SEQ ID NO: 3, which        has glucoamylase activity.-   [7] The variant of paragraph 6, wherein the parent glucoamylase has    at least 65%, at least 70%, at least 75%, at least 80%, at least    85%, at least 90%, at least 95%, at least 96%, at least 97%, at    least 98%, at least 99% or 100% sequence identity to the polypeptide    of SEQ ID NO: 3.-   [8] The variant of paragraph 6 or 7, wherein the parent glucoamylase    is encoded by a polynucleotide that hybridizes under low stringency    conditions, medium stringency conditions, medium-high stringency    conditions, high stringency conditions, or very high stringency    conditions with (i) the mature polypeptide coding sequence of SEQ ID    NO: 1 or (ii) the full-length complement of (i).-   [9] The variant of any of paragraphs 6-8, wherein the parent    glucoamylase is encoded by a polynucleotide having at least 65%, at    least 70%, at least 75%, at least 80%, at least 85%, at least 90%,    at least 95%, at least 96%, at least 97%, at least 98%, at least    99%, or 100% sequence identity to the mature polypeptide coding    sequence of SEQ ID NO: 1.-   [10] The variant of any of paragraphs 1-9, wherein the number of    substitutions is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6,    7, 8, 9 or 10 substitutions.-   [11] The variant of any of the preceding paragraphs, comprising a    substitution or deletion in at least one position selected from    position 65, 327, 501, 504 of SEQ ID NO: 3.-   [12] The variant of any of the preceding paragraphs, wherein the    variant comprises or consists of one or more substitutions selected    from the group consisting of T65A, Q327F, E501V, Y504T, Y504*.-   [13] The variant of any of the preceding paragraphs, wherein the    variant comprises at least one of the following substitutions or    combinations of substitutions:-   T65A; or-   Q327F; or-   E501V; or-   Y504T; or-   Y504*; or-   T65A+Q327F; or-   T65A+E501V; or-   T65A+Y504T; or-   T65A+Y504*; or-   Q327F+E501V; or-   Q327F+Y504T; or-   Q327F+Y504*; or-   E501V+Y504T; or-   E501V+Y504*; or-   T65A+Q327F+E501V; or-   T65A+Q327F+Y504T; or-   T65A+E501V+Y504T; or-   Q327F+E501V+Y504T; or-   T65A+Q327F+Y504*; or-   T65A+E501V+Y504*; or-   Q327F+E501V+Y504*; or-   T65A+Q327F+E501V+Y504T; or-   T65A+Q327F+E501V+Y504*.-   14. The variant of any of the paragraphs 1-13, wherein the variant    comprises at least one of the following combinations of    substitutions:-   E501V+Y504T;-   T65A+K161S;-   T65A+Q405T;-   T65A+Q327W;-   T65A+Q327F;-   T65A+Q327Y;-   P11F+T65A+Q327F;-   R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F;-   P2N+P4S+P11F+T65A+Q327F;-   P11F+D26C+K33C+T65A+Q327F;-   P2N+P4S+P11F+T65A+Q327W+E501V+Y504T;-   R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F;-   P11F+T65A+Q327W;-   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T;-   P11F+T65A+Q327W+E501V+Y504T;-   T65A+Q327F+E501V+Y504T;-   T65A+S105P+Q327W;-   T65A+S105P+Q327F;-   T65A+Q327W+S364P;-   T65A+Q327F+S364P;-   T65A+S103N+Q327F;-   P2N+P4S+P11F+K34Y+T65A+Q327F;-   P2N+P4S+P11F+T65A+Q327F+D445N+V447S;-   P2N+P4S+P11F+T65A+1172V+Q327F;-   P2N+P4S+P11F+T65A+Q327F+N502*;-   P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E;-   P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S;-   P2N+P4S+P11F+T65A+Q327F+S377T;-   P2N+P4S+P11F+T65A+V325T+Q327W;-   P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T;-   P2N+P4S+P11F+T65A+1172V+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T;-   P2N+P4S+P11F+D26N+K34Y+T65A+Q327F;-   P2N+P4S+P11F+T65A+Q327F+1375A+E501V+Y504T;-   P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T;-   P2N+P4S+T10D+T65A+Q327F+E501V+Y504T;-   P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T;-   K5A+P11F+T65A+Q327F+E501V+Y504T;-   P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T;-   P2N+T10E+E18N+T65A+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N;-   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A;-   P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T;-   P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T;-   P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A;-   P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*;-   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T;-   K5A+P11F+T65A+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A;-   P2N+P4S+P11F+T65A+V79A+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+V79G+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+V79I+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+V79L+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+V79S+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T;-   S255N+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or-   P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T.-   [15] The variant of any of paragraphs 1-14, which has an improved    property relative to the parent, wherein the improved property is    reduced sensitivity to protease degradation.-   [16] The variant of any of paragraphs, 1-15, which has an improved    property relative to the parent, wherein the improved property is    improved thermostability.-   [17] A variant glucoamylase catalytic domain comprising a    substitution at one or more positions corresponding to positions 10,    11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112, 161,    172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370,    375, 377, 405, 445, 447, 460, 463, 465, 468 of the polypeptide of    SEQ ID NO: 3, wherein the variant has glucoamylase activity.-   [18] The variant glucoamylase catalytic domain of paragraph 17    selected from the group consisting of:    -   (a) a catalytic domain having at least 65% sequence identity to        amino acids 30 to 494 of SEQ ID NO: 2;    -   (b) a catalytic domain encoded by a polynucleotide that        hybridizes under medium stringency conditions with (i)        nucleotides 88 to 1482 of SEQ ID NO: 1 or (ii) the full-length        complement of (i);    -   (c) a catalytic domain encoded by a polynucleotide having at        least 65% sequence identity to (i) nucleotides 88 to 1482 of SEQ        ID NO: 1; and-   wherein the catalytic domain has glucoamylase activity.-   [19] The polypeptide of paragraph 18, further comprising a linker    and a carbohydrate binding domain.-   [20] A composition comprising the polypeptide of any of paragraphs    1-19.-   [21] A composition according to paragraph 20, comprising an    alpha-amylase and a polypeptide of any of paragraphs 1-19.-   [22] A use of a polypeptide of any of paragraphs 1-19 for production    of syrup and/or a fermentation product.-   [23] The use according to paragraph 22, wherein the starting    material is gelatinized or un-gelatinized starch-containing    material.-   [24] A use of a polypeptide of any of paragraphs 1-19 for brewing.-   [25] A process of producing a fermentation product from    starch-containing material comprising the steps of:    -   (a) liquefying starch-containing material in the presence of an        alpha amylase;    -   (b) saccharifying the liquefied material; and    -   (c) fermenting with a fermenting organism;        wherein step (a) and/or step (b) is carried out using at least a        glucoamylase of any of paragraphs 1-19.-   [26] A process of producing a fermentation product from    starch-containing material, comprising the steps of:    -   (a) saccharifying starch-containing material at a temperature        below the initial gelatinization temperature of said        starch-containing material; and    -   (b) fermenting with a fermenting organism,        wherein step (a) is carried out using at least a glucoamylase of        any of paragraphs 1-19.-   [27] An isolated polynucleotide encoding the polypeptide of any of    paragraphs 1-19.-   [28] A nucleic acid construct or expression vector comprising the    polynucleotide of paragraph 27 operably linked to one or more    control sequences that direct the production of the polypeptide in    an expression host.-   [29] A recombinant host cell comprising the polynucleotide of    paragraph 27 operably linked to one or more control sequences that    direct the production of the polypeptide.-   [30] A method of producing the polypeptide of any of paragraphs    1-19, comprising:    -   (a) cultivating a cell, which in its wild-type form produces the        polypeptide, under conditions conducive for production of the        polypeptide; and    -   (b) recovering the polypeptide.-   [31] A method of producing a polypeptide of any of paragraphs 1-19,    comprising:    -   (a) cultivating the host cell of paragraph 29 under conditions        conducive for production of the polypeptide; and    -   (b) recovering the polypeptide.-   [32] A nucleic acid construct comprising the polynucleotide of    paragraph 27.-   [33] An expression vector comprising the polynucleotide of paragraph    27.-   [34] A host cell comprising the polynucleotide of paragraph 27.

The invention claimed is:
 1. A glucoamylase variant having improvedthermostability, comprising a substitution at a positon corresponding toposition 65 of the polypeptide of SEQ ID NO: 3,wherein the variant hasglucoamylase activity and wherein the variant has at least 90% sequenceidentity to the polypeptide of SEQ ID NO:
 3. 2. The variant of claims 1,wherein the variant has at least at 95% sequence identity to SEQ ID NO:3.
 3. The variant of claim 1, wherein the variant has at least 97%sequence identity to the polypeptide of SEQ ID NO:
 3. 4. The variant ofclaim 1, wherein the variant has at least 98% sequence identity to SEQID NO:
 3. 5. The variant of claim 1, wherein the variant has at least99% sequence identity to SEQ ID NO:
 3. 6. The variant of claim 1,wherein the variant has a number of substitutions in the amount of 1-20.7. The variant of claim 1, further comprising one or more substitutionor deletion in at least one position selected from position 327, 501,and 504 of SEQ ID NO:
 3. 8. The variant of claim 1, wherein the variantfurther comprises or consists of one or more substitutions selected fromthe group consisting of T65A, Q327F, E501V, Y504T, and Y504*.
 9. Thevariant of claim 1, wherein the variant comprises at least one of thefollowing alterations or combinations of alterations: T65A; or T65A+Q327F; or T65A +E501V; or T65A +Y504T; or T65A +Y504*; or T65A +Q327F+E501V; or T65A +Q327F +Y504T; or T65A +E501V +Y504T; or T65A +Q327F+Y504*; or T65A +E501V +Y504*; or T65A +Q327F +E501V +Y504T; or T65A+Q327F +E501V +Y504*.
 10. The variant of claim 1, wherein the variantcomprises one of the following combinations of substitutions: T65A+K161S; T65A +Q405T; T65A +Q327W; T65A +Q327F; T65A +Q327Y; P11F +T65A+Q327F; R1 K +D3W +K5Q +G7V +N8S +T1OK +P11S +T65A +Q327F; P2N +P4S+P11F +T65A +Q327F; P11F +D26C +K33C +T65A +Q327F; P2N +P4S +P11F +T65A+Q327W +E501V +Y504T; R1E +D3N +P4G +G6R +G7A +N8A +T10D+P11D +T65A+Q327F; P11F +T65A +Q327W; P2N +P4S +P11F +T65A +Q327F +E501V +Y504T;P11F +T65A +Q327W +E501V +Y504T; T65A +Q327F +E501V +Y504T; T65A +S105P+Q327W; T65A +S105P +Q327F; T65A +Q327W +S364P; T65A +Q327F +S364P; T65A+S103N +Q327F; P2N +P4S +P11F +K34Y +T65A +Q327F; P2N +P4S +P11F +T65A+Q327F +D445N +V447S; P2N +P4S +P11F +T65A +I172V +Q327F; P2N +P4S +P11F+T65A +Q327F +N502*; P2N +P4S +P11F +T65A +Q327F +N502T +P563S +K571E;P2N +P4S +P11F +R31S +K33V +T65A +Q327F +N564D +K571S; P2N +P4S +P11F+T65A +Q327F +S377T; P2N +P4S +P11 F +T65A +V325T+Q327W; P2N +P4S +P11 F+T65A +Q327F +D445N +V447S +E501V +Y504T; P2N +P4S +P11F +T65A +1172V+Q327F +E501V +Y504T; P2N +P4S +P11F +T65A +Q327F +S377T +E501V +Y504T;P2N +P4S +P11F +D26N +K34Y +T65A +Q327F; P2N +P4S +P11F +T65A +Q327F+1375A +E501V +Y504T; P2N +P4S +P11F +T65A +K218A +K221D +Q327F +E501V+Y504T; P2N +P4S +P11F +T65A +S103N +Q327F +E501V +Y504T; P2N +P4S +T1OD +T65A +Q327F +E501V +Y504T; P2N +P4S +F12Y +T65A +Q327F +E501V+Y504T; K5A +P11F +T65A +Q327F +E501V +Y504T; P2N +P4S +T1OE +E18N +T65A+Q327F +E501V +Y504T; P2N +T1OE +E18N +T65A +Q327F +E501V +Y504T; P2N+P4S +P11F +T65A +Q327F +E501V +Y504T +T568N; P2N +P4S +P11 F +T65A+Q327F +E501V +Y504T +K524T +G526A; P2N +P4S +P11 F +K34Y +T65A +Q327F+D445N +V447S +E501V +Y504T; P2N +P4S +P11F +R31S +K33V +T65A +Q327F+D445N +V447S +E501V +Y504T; P2N +P4S +P11F +D26N +K34Y +T65A +Q327F+E501V +Y504T; P2N +P4S +P11F +T65A +F80* +Q327F +E501V +Y504T; P2N +P4S+P11F +T65A +K112S +Q327F +E501V +Y504T; P2N +P4S +P11F +T65A +Q327F+E501V +Y504T +T516P +K524T +G526A; P2N +P4S +P11F +T65A +Q327F +E501V+N502T +Y504*; P2N +P4S +P11F +T65A +Q327F +E501V +Y504T; P2N +P4S +P11F+T65A +S103N +Q327F +E501V +Y504T; K5A +P11F +T65A +Q327F +E501V +Y504T;P2N +P4S +P11F +T65A +Q327F +E501V +Y504T +T516P +K524T +G526A; P2N +P4S+P11F +T65A +V79A +Q327F +E501V +Y504T; P2N +P4S +P11F +T65A +V79G+Q327F +E501V +Y504T; P2N +P4S +P11 F +T65A +V791 +Q327F +E501V +Y504T;P2N +P4S +P11 F +T65A +V79L +Q327F +E501V +Y504T; P2N +P4S +P11F +T65A+V79S +Q327F +E501V +Y504T; P2N +P4S +P11 F +T65A +L72V +Q327F +E501V+Y504T; P2N +P4S +P11 F +T65A +E74N +V79K +Q327F +E501V +Y504T; P2N +P4S+P11F +T65A +G220N +Q327F +E501V +Y504T; P2N +P4S +P11F +T65A +Y245N+Q327F +E501V +Y504T; P2N +P4S +P11F +T65A +Q253N +Q327F +E501V +Y504T;P2N +P4S +P11F +T65A +D279N +Q327F +E501V +Y504T; P2N +P4S +P11F +T65A+Q327F +S359N +E501V +Y504T; P2N +P4S +P11F +T65A +Q327F +D370N +E501V+Y504T; P2N +P4S +P11F +T65A +Q327F +V460S +E501V +Y504T; P2N +P4S +P11F +T65A +Q327F +V460T +P468T +E501V +Y504T; P2N +P4S +P11F +T65A +Q327F+T463N +E501V +Y504T; P2N +P4S +P11F +T65A +Q327F +S465N +E501V +Y504T;or P2N +P4S +P11F +T65A +Q327F +T477N +E501V +Y504T.
 11. The variant ofclaim 1, which has an improved property relative to a parent, whereinthe improved property is reduced sensitivity to protease degradation.12. The variant of claim 1, which has an improved property relative to aparent, wherein the improved property is improved thermostability.
 13. Avariant glucoamylase catalytic domain comprising a substitution at aposition corresponding to position 65 of the polypeptide of SEQ ID NO:3, wherein the variant has glucoamylase activity.
 14. A compositioncomprising the polypeptide of claim
 1. 15. A composition according toclaim 14, comprising an alpha-amylase.
 16. A process of producing afermentation product from starch-containing material comprising thesteps of: (a) liquefying starch-containing material in the presence ofan alpha amylase; (b) saccharifying the liquefied material; and (c)fermenting with a fermenting organism; wherein step (a) and/or step (b)is carried out using at least a glucoamylase of claim
 1. 17. A processof producing a fermentation product from starch-containing material,comprising the steps of: (a) saccharifying starch-containing material ata temperature below the initial gelatinization temperature of saidstarch-containing material; and (b) fermenting with a fermentingorganism, wherein step (a) is carried out using at least a glucoamylaseof claim 1.